HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Farrell, H. M. Articles by Swaisgood, H. E. Search for Related Content PubMed PubMed Citation Articles by Farrell, H. M., Jr. Articles by Swaisgood, H. E.

Nomenclature of the Proteins of Cows’ Milk—Sixth Revision

¹ US Department of Agriculture, Eastern Regional Research Center, Wyndmoor, PA 19038
² Department of Food Science, California Polytechnic State University, San Luis Obispo 93407
³ Gala Design, Middleton, WI 53562
⁴ Department of Microbiology, School of Medicine, University of Iowa, Iowa City 52240
⁵ Fonterra Research Centre, Palmerston North, New Zealand
⁶ Department of Animal Science, University of Kentucky, Lexington 40546
⁷ Masterfoods USA, Burr Ridge, IL 60527
⁸ Department of Animal Science, McGill University, Sainte Anne de Bellevue, PQ, Canada H9X 3V9
⁹ Department of Food Science, North Carolina State University, Raleigh 27695

Corresponding author: H. M. Farrell, Jr.; e-mail: hfarrell{at}errc.ars.usda.gov.

	ABSTRACT

TOP ABSTRACT INTRODUCTION THE CASEINS THE WHEY PROTEINS REFERENCES

 
This report of the American Dairy Science Association Committee on the Nomenclature, Classification, and Methodology of Milk Proteins reviews changes in the nomenclature of milk proteins necessitated by recent advances of our knowledge of milk proteins. Identification of major caseins and whey proteins continues to be based upon their primary structures. Nomenclature of the immunoglobulins consistent with new international standards has been developed, and all bovine immunoglobulins have been characterized at the molecular level. Other significant findings related to nomenclature and protein methodology are elucidation of several new genetic variants of the major milk proteins, establishment by sequencing techniques and sequence alignment of the bovine caseins and whey proteins as the reference point for the nomenclature of all homologous milk proteins, completion of crystallographic studies for major whey proteins, and advances in the study of lactoferrin, allowing it to be added to the list of fully characterized milk proteins.

Key Words: milk protein • structure • nomenclature • review

Abbreviation key: C = constant, EIMS = electrospray ionization MS, H = heavy chain, HSA = human SA, L = light chain, LF = lactoferrin, MS = mass spectroscopy, NMR = nuclear magnetic resonance, SA = serum albumin, V = variable

	INTRODUCTION

TOP ABSTRACT INTRODUCTION THE CASEINS THE WHEY PROTEINS REFERENCES

 
The initial report of the American Dairy Science Association Committee on the Nomenclature, Classification, and Methodology of Milk Proteins (Jenness et al., 1956) was an attempt to clarify the nomenclature of milk proteins by "presenting a summary of preferred usage and by showing the relationship between the individual proteins, which had been isolated, and the classical fractions." Subsequently, this Committee has published a revision of milk protein nomenclature approximately every 5 to 10 yr to summarize more recent findings (Table 1) and to suggest changes in nomenclature where appropriate. The intent of this Committee is to suggest a flexible nomenclature system that allows for incorporation of new discoveries rather than to suggest prematurely a rigid system of nomenclature. Since the last report of this Committee (Eigel et al., 1984), the most significant findings related to nomenclature and protein methodology are as follows.

• Elucidation of the primary structures of many proteins associated with the fat globule membrane The rapid progress in this latter area and the importance of these membrane-associated proteins to milk led to a separate review of these proteins. In a review sponsored by this Committee, the current nomenclature for proteins associated with the milk fat globule membrane has been summarized (Mather, 2000). This report will deal only with the proteins of skim milk. In addition, in keeping with past practice, we will not elaborate on enzymes associated with whole milk or skim milk. These enzymes have been reviewed elsewhere (Shahani et al., 1973; Jenness, 1974; Swaisgood, 1995).
  
• New genetic variants and milk proteins The discovery of new genetic variants has contributed to the establishment of a broader base of information on milk proteins (Formaggioni et al., 1999; Ng-Kwai-Hang and Grosclaude, 2003). The widespread application of molecular biology techniques to probe the "milk protein genome" has brought about an interesting dilemma in the study of milk proteins. Because of the built-in redundancy in the genetic code, it is possible to have a multiplicity of, e.g., -CN genes CSN3, that will code for the same protein (-CN). Note that the designation at the CSN3 locus relates to the DNA sequence or genotype, and CN denotes the phenotypic expression of the gene, the protein molecule. For example, Prinzenberg et al. (1999) demonstrated the presence of a silent mutation in the CSN3*A locus; here, the change of CCA to CCG still codes for proline at residue 152. This locus is termed CSN3*A₁, but the protein remains -CN A. As an interesting aside, for single codon changes in the 3 base code, there could be up to 575 silent changes in the DNA, yet the expressed protein would remain -CN A. Because this Committee has focused on protein methodologies and sequences, it is the intent to assign letter symbols only to those proteins that are chemically different from the reference protein or a known member of the family. In this respect, no matter how many codon changes there are in the DNA, if the protein is chemically identical to a member of the family, then it would still be, e.g., -CN A.
  
• Establishment by sequencing techniques and sequence alignment of the bovine CN and whey proteins as the reference point for the nomenclature of all homologous milk proteins. In this regard, several free on-line protein databases have been established.
  
• In addition to serving as on-going information repositories, these web sites contain convenient programs for the calculation of theoretical protein parameters, sequence alignments, and structural predictions. The following list contains some of these sites, but others are equally valuable.

  National Center of Biotechnology Information: www.ncbi.nih.gov/database
  

  The Swiss Protein and European Molecular Biology Site (ExPASy): www.expasy.org
  

  Georgetown University Protein Information Resource: http://pir.georgetown.edu
  

  Protein Science Site: http://www.proteinsociety.org/
  

  A new database for protein researchers is being constructed under an NIH contract. It will combine the current Swiss-Prot, Trembl, and PIR resources into a single searchable database. The new resources will be called United Protein Data Base or UniProt, and, when completed (~2005), its web site will be www.uniprot.org. The current sites will continue to function as conduits for the new database.
  

  The European Bioinformatics Institute: www.ebi.ac.uk
  

  Comparative Ig Web site: www.medicine.uiowa.edu/CIgW
  

  IMGTIg web site: http://imgt.cines.fr/
  

  
  
• Characterization of all bovine Ig at the molecular levels, including mapping of the heavy chain (H) constant (C) region locus, defining the mechanism of variable heavy- and lambda- chain repertoire development, and establishing international standards for nomenclature (www.medicine.uiowa.edu/CIgW).
  
• Crystallographic studies have been completed on the major whey proteins as detailed subsequently. Crystal structures for viewing are available through the Protein Data Bank at www.rcsb.org/pdb. In the absence of crystallographic data for the CN, working 3-D molecular models have been presented as a point of departure for future studies of CN structure (Kumosinski et al., 1994). These models are available at www.arserrc.gov/dpp/casein.htm.
  
• Progress in the sequence analysis of lactoferrin (LF) allows this protein to be reviewed for the first time by this Committee.
  

	THE CASEINS

TOP ABSTRACT INTRODUCTION THE CASEINS THE WHEY PROTEINS REFERENCES

 
Caseins in milk of the genus Bos were defined originally by this Committee (Jenness et al., 1956) as those phosphoproteins that precipitate from raw skim milk by acidification to pH 4.6 at 20°C. In a subsequent report (Whitney et al., 1976), the Committee differentiated CN according to their relative electrophoretic mobility in alkaline polyacrylamide or starch gels containing urea with or without mercaptoethanol. In the previous report, we recommended that use of electrophoresis as a basis for classification be dropped and that CN be identified according to the homology of their primary structures (amino acid sequences) into the following families: _s1-, _s2-, ß-, and -CN. This recommendation is affirmed, and researchers are requested to refrain from assigning specific genetic variant letters to new variants until their sequence homology can be established. Individual members of these families still can be identified by gel electrophoretic techniques, some of the more effective of which are suggested in the monograph by this Committee (Swaisgood et al., 1975).

_S1-CN
The _Sl-CN family, which constitutes up to 40% of the CN fraction in bovine milk, consists of one major and one minor component. Both proteins are single-chain polypeptides with the same amino acid sequence established by Mercier et al. (1971) and Grosclaude et al. (1973) and differ only in their degree of phosphorylation. The minor component contains one additional phosphorylated serine residue at position 41 (Eigel et al., 1984). The reference protein for this family is _S1-CN B-8P, a single-chain protein with no cysteinyl residues. It consists of 199 amino acid residues: Asp₇, Asn₈, Thr₅, Ser₈, Ser P₈, Glu₂₅, Gln_l4, Pro_l7, Gly₉, Ala₉, Val₁₁, Met₅, Ile₁₁, Leu_l7, Tyr_l0, Phe₈, Lys_l4, His₅, Trp₂, and Arg₆ with a calculated molecular weight of 23,615 (Mercier et al., 1971). Its primary sequence is given in Figure 1; its ExPASy entry name and file number are CAS1_Bovin and P02662, respectively. Since the last nomenclature report (Eigel et al., 1984), 3 new genetic variants of _S1-CN have been identified. They are _s1-CN F (Erhardt, 1993), which was found in German Black and White cattle; _S1-CN G (Mariani et al., 1995) discovered in Italian Brown cows; and the H variant (Mahé et al. 1999). Hence, this family of proteins is currently known to consist of variant A found in Holstein Friesians, Red Holsteins, and German Red cattle (Ng-Kwai-Hang et al., 1984; Grosclaude, 1988; Erhardt, 1993); variant B, which is the predominant variant in Bos taurus (Eigel et al., 1984); variant C in Bos indicus and Bos grunniens (Eigel et al., 1984); variant D in various breeds in France (Grosclaude, 1988) and Italy (Mariani and Russo, 1975) as well as in Jerseys in The Netherlands (Corradini, 1969); and variant E in Bos grunniens (Grosclaude et al., 1976) in addition to the new variants F, G, and H.

View larger version (28K): [in this window] [in a new window]   Figure 1. The primary structure of S1-CN B-8P (Mercier et al., 1971; Grosclaude et al., 1973; Stewart et al., 1984; Nagao et al., 1984; Koczan et al., 1991). Amino acid deletion or substitutions for genetic variants A, C, D, E, F, G, and H, respectively, are indicated in Table 2. Sites of post-translational phosphorylation (SeP) are indicated in italicized, boldface type. The underline indicates the location of another phosphorylation site in a minor species of this protein (S1-CN B9-P).

Since the discovery of genetic variants, attempts have been made to correlate milk characteristics or milk production with the genotype. However, the correlations obtained have not been straightforward, in part because of differences in the parameters used. For example, the _S1-CN BB phenotype has been correlated with higher milk yields and, thus, higher protein yield over the lactation, (Ng-Kwai-Hang et al., 1984; Aleandri et al., 1990; Sang et al., 1994), but the same phenotype has also been correlated with lower protein concentration in milk (Ng-Kwai-Hang et al., 1986, 1992). It appears that cows carrying the G allele produce less _S1-CN and more of the other caseins (Mariani et al., 1995). For example, homozygous (GG) cows produce 55% less _S1-CN.

Because of the 13-amino acid residue deletion, the A variant’s characteristics are most different from the other variants (Farrell et al., 1988). Thus, most of the hydrophobic residues in the N-terminal region are eliminated, including the Phe-Phe-Val sequence that is cleaved by chymosin during cheese ripening (Mulvihill and Fox, 1979). Hence, _S1-CN A is similar to the peptide _S1-I corresponding to _S1-CN (f25-199), which has a reduced hydrophobicity (Creamer et al., 1982) and does not aggregate as extensively in the presence of calcium (Kaminogawa et al., 1980). The changes in curd rheology that occur with this proteolysis of the B variant are consistent with the observation that soft curds are formed with milks containing _S1-CN A (Sadler et al., 1968).

Comparison of the properties of variants B and C has indicated that _S1-CN C self-associates more strongly (Schmidt, 1970; Swaisgood, 1973), and cheeses made from milks containing the latter form a tougher curd (Sadler et al., 1968).

The distinct regions of anionic clusters and hydrophobicity evident in the primary structure are suggestive of the formation of hydrophobic and polar domains (Swaisgood, 1982, 1992) and are consistent with observed physical-chemical properties, such as the strong dependence of association on concentration, pH, ionic strength, and ion binding. The characteristics and significance of calcium ion binding to the anionic clusters are well known, but it has also been found that Zn²⁺ (Singh et al., 1989) and Fe (III) (Reddy and Mahoney, 1991) bind at these sites. The effect of these interactions on micelle structure and stability is not known.

_S2-CN
The _S2-CN family, which constitutes up to 10% of the CN fraction in bovine milk, consists of 2 major and several minor components exhibiting varying levels of post-translational phosphorylation (Swaisgood, 1992) and minor degrees of intermolecular disulfide bonding (Rasmussen et al., 1992). The predominant forms in bovine milk contain an intramolecular disulfide bond and differ only in their degree of phosphorylation. The reference protein for this family is _S2-CN A-11P, a single-chain polypeptide with an internal disulfide bond. It consists of 207 amino acid residues: Asp₄, Asn_l4, Thr_l5, Ser₆, Ser P₁₁, Glu₂₄, Gln₁₆, Pro₁₀, Gly₂, Ala₈, Cys₂, Val₁₄, Met₄, Ile₁₁, Leu₁₃, Tyr₁₂, Phe₆, Lys₂₄, His₃, Trp₂, and Arg₆ with a calculated formula molecular weight of 25,226. The primary structure of this protein is given in Figure 2; its ExPASy entry name and file number are CAS2_Bovin and P02663, respectively. The secondary structure of _S2-CN has recently been studied by CD and FTIR spectroscopies (Hoagland et al., 2001).

View larger version (30K): [in this window] [in a new window]   Figure 2. The primary structure of the S2-CN A-11P (Brignon et al., 1977; Mahé and Grosclaude, 1982; Stewart et al., 1987; Groenen et al., 1993). Seryl residues (SeP) identified as phosphorylated in S2-CN A-11P are indicated in italicized, boldface type. Residues that have been determined to be partially phosphorylated or that potentially may be phosphorylated according to CN kinase specificity are underlined. Amino acid deletions or substitutions for genetic variants are given in Table 2.

The primary structure of _S2-CN A-11P (Figure 2), reported by Brignon et al. (1977), has been changed to Gln at position 87 rather than Glu, as indicated by cDNA sequencing (Stewart et al., 1987) and genomic DNA sequencing (Groenen et al., 1993). The _s2-CN signal peptide is composed of 15 amino acid residues, making the pre-form 222 amino acid residues in length. The D variant differs from _S2-CN A by the deletion of 9 amino acid residues from positions 51 to 59. However, the genomic DNA sequence does not reveal a deletion, but rather a substitution, suggesting that the amino acid sequence deletion is caused by the skipping of exon VIII, a 27-nucleotide sequence that encodes amino acid residues 51 to 59 (Bouniol et al., 1993). As shown in Table 2, the C variant differs from the A variant at positions 33, 47, and 130 (Mahé and Grosclaude, 1982). As the specific sites of mutation resulting in _S2 -CN B have not been identified, as shown in Table 2. Because of the progress made on this protein, following the elucidation of its sequence, _S2-CN will be reviewed in more detail in this report. Post-translational phosphorylation, primarily at seryl residues, results in the incorporation of 10 to 13 phosphate moeities. According to the specificity of CN kinase, phosphorylation occurs at Ser/Thr residues in the sequence Ser/Thr-X-Glu/SerP/Asp; however, the sequence SerX-Glu/SerP is heavily favored (Mercier, 1981). Only seryl residues are phosphorylated in _S2-CN A-11P, but Thr-66 was partially phosphorylated in _S2-CN C (Mahe and Grosclaude, 1982). Those residues known to be phosphorylated in _S2-CN A-11P are indicated by boldface italics in the figure. The underlined residues indicate potential sites of phosphorylation suggested by the enzyme specificity. It should be noted that Thr-47 in _S2-CN C is a potential phosphorylation site.

Another post-translational change that occurs with this protein is the formation of disulfide bonds. The 2 cysteinyl residues of this protein participate in both intramolecular and intermolecular disulfide bonds (Rasmussen et al., 1992, 1994). The protein exists predominantly as a monomer (>85%) with a disulfide bond between Cys residues 36 and 40 (Rasmussen et al., 1994) or as a dimer with both parallel and antiparallel disulfide bonds (Rasmussen et al., 1992). Therefore, 2 types of dimers are found: one fraction with residues 36 and 40 in one chain linked to residues 36 and 40, respectively, in the other chain. But, in another fraction, residues 36 and 40 are linked to residues 40 and 36, respectively, in the other chain. These results suggest that the formation of these bonds is not important to any structure required by this protein for its interaction with other CN.

_S2-Casein is the most hydrophilic of all caseins as a result of the 3 clusters of anionic groups composed of phosphoseryl and glutamyl residues. Although relatively hydrophobic, the C-terminal 47 residues carry a net positive charge (about +9.5) at the pH of milk (Swaisgood, 1992). On the other hand, the more hydrophilic N-terminal 68 residues contain 2 anionic clusters and exhibit a net charge of about –21 at the prevalent pH of milk. Hence, the primary structure of _S2-CN can be represented by 4 domains: an N-terminal hydrophilic domain with anionic clusters, a central hydrophobic domain, followed by another hydrophilic domain with anionic clusters, and finally a C-terminal positively charged hydrophobic domain (Swaisgood, 1992). This structure is consistent with an association behavior that is very dependent on ionic strength (Snoeren et al., 1980). The association appears to be strongest around an ionic strength of 0.2 M, with dissociation occurring in lower salt because of electrostatic repulsion and also in higher salt because of suppression of electrostatic attraction, thus, reflecting the contributions of both hydrophobic interactions and electrostatic attraction.

The number of anionic clusters and the hydrophilic nature is also reflected in calcium-binding properties of _S2-CN. For example, the latter protein is more sensitive to Ca²⁺ than _S1-CN (Toma and Nakai, 1973), with almost complete precipitation occurring in 2 mM Ca²⁺ for _S2-CN at pH 7; whereas, precipitation of _S1-CN requires 6 mM Ca²⁺ (Aoki et al., 1985). These properties also led to a method for fractionation of _S2-CN from other caseins by precipitation from propan-l-ol solutions (Vreeman and van Riel, 1990). Solubility in this solvent is governed by electrostatic interactions that are most prevalent in _S2-CN.

_S2-Casein appears to be readily susceptible to proteolysis as assessed by the activities of chymosin and plasmin toward the protein. Chymosin activity was observed in the regions of residues 88 to 98 and 164 to 180, but its primary cleavage occurred at Phe 88-Tyr 89 (McSweeney et al., 1994). These 2 regions are, respectively, at the edge of the central hydrophobic domain or in the first part of the cationic hydrophobic C-terminal domain. Plasmin activity released a number of peptides, including the N-terminal 21 to 24 residues of the initial hydrophilic domain containing one of the anionic clusters (Le Bars and Grippon, 1989; Visser et al., 1989). In agreement with plasmin specificity, mostly Lys-X bonds were cleaved at varying rates (Lys residues 21, 24, 149, 150, 181, 188, and 197). In addition to the shorter N-terminal peptides, a major peptide released was _S2-CN (fl51-207) (Le Bars and Grippon, 1989). In this regard, it is interesting to note that recently _S2-CN (fl65-203) was isolated from milk and shown to have antibacterial activity (Zucht et al., 1995).

ß-CN
The ß-CN family, which constitutes up to 45% of the casein of bovine milk is quite complex because of the action of the native milk protease plasmin (Eigel et al., 1984). Plasmin cleavage leads to formation of ₁-, ₂-. and ₃-CN, which are actually fragments of ß-CN consisting of residues 29-209, 106-209, and 108-209. In addition, polypeptides previously called proteose peptone components 5, 8-fast, and 8-slow are fragments of ß-CN, which represent residues 1-105 or 1-107, 1-28, and 29-105, respectively. The reference protein for this family, ß-CN A²-5P is a single-polypeptide chain with no Cys residues containing 209 residues. It consists of Asp₄, Asn₅, Thr₉, Ser₁₁, Ser P₅, Glu_l9, Gln₂₀, Pro₃₅, Gly₅, Ala₅, Val₁₉, Met₆, Ile_l0, Leu₂₂, Tyr₄, Phe₉, Lys₁₁, His₅, Trp₁, and Arg₄ with a calculated molecular weight of 23,983. The most common variant used as reference is variant A²; its ExPASy entry name and file number are CASB_Bovin and P02666, respectively. The A² variant has been chemically sequenced (Ribadeau-Dumas et al., 1972) and sequenced from its cDNA (Jimenez-Flores et al., 1987; Stewart et al., 1987) and its gene (Bonsing et al., 1988). The ß-CN signal peptide is composed of 15 amino acid residues, making the pre-form 224 amino acids in length.

The sequence shown for ß-CN A² in Figure 3 is that as corrected by 2 groups (Yan and Wold, 1984; Carles et al., 1988). It differs from the original sequence (Eigel et al., 1984) in 4 places: Gln117Glu, Pro 137 and Leu 138 are inverted, Glu175Gln, and Gln195Glu. The changes at residues 117 and 175 are confirmed by both groups and by gene sequencing. The inversion of residues 137 and 138 are not in agreement with cDNA sequencing data (Jimenez-Flores et al., 1987), which is in accordance with the original data. However the Leu-Pro substitution is a one base change, and mutations could occur and not be observed by HPLC-mass spectroscopy (MS) of peptides or by electrophoresis of the proteins. The weight here is, however, given to the 2 independent protein-sequencing reports. In a similar fashion, the change at 195 is not in agreement with the cDNA results, but, in this case, 3 lines of evidence support the occurrence of only Glu at residue 195. They include the following:

View larger version (29K): [in this window] [in a new window]   Figure 3. Primary structure of Bos ß-CN A2-5P (Ribadeau-Dumas et al., 1972; Grosclaude et al. 1973). The amino acid residues corresponding to the mutational differences in the genetic variants, A1, A3, B, C, D, E, F, G, H, and I are indicated in Table 2. Sites of post-translational phosphorylation (SeP) are indicated in italicized, boldface type. The arrows indicate the points of attack by plasmin responsible for ß-CN fragments (-CN and proteose peptones) present in milk.

• the 2 protein sequencing corrections noted previously;
  
• the invariance on electrophoresis of ß-CN (f108-209) from A¹, A², and A³ genetic variants (Groves, 1969); and
  
• the purification from cheese of a bitter peptide ß-CN (f193-209) whose sequence is identical to the chemically corrected sequences (Gouldsworthy et al., 1996).
  

The previous report of Eigel et al. (1984) described 7 genetic variants. Since that revision, 3 new variants have been identified by sequence: ß-CN F, previously called ß-CN X (Visser et al., 1995); ß-CN G (Dong and Ng-Kwai-Hang, 1998); and ß-CN H (Han et al., 2000). The amino acid substitutions giving rise to all variants of ß-CN are given in Table 2. In addition, Chung et al. (1995) identified variant A⁴ in native Korean cattle using electrophoresis only; its substitutions in the A² reference protein are unknown.

Visser et al. (1995) identified ß-CN F, which contains the A¹ substitution and Leu for Pro at residue 152. ß-casein F was separated by preparative reverse-phase HPLC. The main differential peaks representing the 114 to 169 fragments of ß-A¹ and ß-X, respectively, were both purified following trypsin digestion, cyanogen bromide cleavage, and separation of the corresponding peptides representing the 145–156 sequence. The presence of Leu residue at position 152 instead of the Pro-152 in ß-CN A¹ was established by fast-atom bombardment MS-MS. In accordance with internationally accepted guidelines for the nomenclature of milk proteins, the new genetic variant has been named ß-CN F-5P. In a similar fashion, Dong and Ng-Kwai-Hang (1998) identified ß-CN G-5P, which is similar to ß-CN A¹ and F, but contains a Leu in place of Pro at either position 137 or 138, depending on the sequence assigned, as the Pro-Leu inversion is controversial. Han et al. (2000) identified ß-CN H, which represents 2 substitutions relative to the corrected reference ß-CN A². These are Arg25 to Cys and Leu88 to Ile. A genetic variant, discovered by Senocq et al. (2002) was also named H; so, it is proposed that the Han variant be termed H¹, and the Senocq variant be termed H². The H² variant differs from the A² variant at 2 known positions (Met93Leu and Gln72Glu) and a substitution of Gln to Glu between residues 114–169. Finally, the I variant was described by Jann et al. (2002); it contains only the Met 93Leu substitution of the H² variant.

ß-Casein is the most hydrophobic of the CN. The N-terminal sequence codes for charged amino acids as well as a phosphoserine cluster. This initial sequence is different from the second half of the molecule, where neutral and hydrophobic amino acid residues abound. Calculation of the net charge at pH 6.6 indicates that the first 21 amino acids would have a net charge of about –11.5, and the C-terminal 21 amino acids (190–209) have no net charge. This molecule presents a high contrast in its sequence, one-tenth of the amino acids at the N-terminus of the protein contain one-third of the total charge, while 75% of the residues at the C-terminal one-tenth consist of hydrophobic amino acids. It is this unusual distribution of amino acids that leads to the release of ß-CN from CN micelles in the cold (Aoki et al., 1990). It is important to mention that no 3-D structure from X-ray crystallography has been reported; however, a computer-generated working 3-D model has been presented (Kumosinski et al., 1993b). Perhaps the difficulty on inducing suitable crystals from this protein is due to its dependence on the environment surrounding it and its propensity for self-association.

Addition of a glycosylation signal in the gene of ß-CN has been reported (Choi and Jimenez-Flores, 1996). This modification of a bovine milk protein is an important aspect of this review because the original gene was generated from ß-CN A¹ with the glycosylation signal, changing from Pro 67 to Ser 67. However, this new variant represents a man-made intervention and does not occur naturally. We suggest that nomenclature of genetic variants induced through molecular biology techniques follow the same mechanisms as those established by this Committee for naturally occurring variants, e.g., a point mutation of ß-CN A¹, Pro67Ser 67.

-CN
The -CN family consists of a major carbohydrate-free component and a minimum of 6 minor components. The 6 minor components, as detected by PAGE in urea with 2-mercaptoethanol (Mackinlay and Wake, 1965; Pujolle et al., 1966; Woychik et al., 1966; Vreeman et al., 1977; Doi et al., 1979), represent varying degrees of phosphorylation and glycosylation.

-casein, as isolated from milk, also occurs in the form of a mixture of disulfide-bonded polymers ranging from dimer to octamers and above (Groves et al., 1992). Beeby (1964) reported the presence of free thiol groups after calcium removal by treatment with ethylenediaminetetraacetate, but other chemical analyses did not confirm this result (Swaisgood et al., 1964). Sodium dodecyl sulfate-gel electrophoresis and physical measurements suggest that the native form of -CN is highly associated both chemically and physically (Swaisgood and Brunner, 1963; Groves et al., 1992; Farrell et al., 1996) and that heat treatment of native -CN results in aggregation caused by free sulfhydryl-disulfide interchange (Groves et al., 1998). Reduction and S-carboxymethylation of -CN followed by heating can result in amyloid (fibrillar) structures (Farrell et al., 2002).

The primary structure of the reference protein of the -CN family is the major carbohydrate-free component of -CN A-1P (Figure 4); its ExPASy entry name and file number are CASK_Bovin and P02668, respectively. It consists of 169 amino acid residues as follows: Asp₄, Asn₈, Thr₁₅, Ser₁₂, Ser P₁, Pyroglu₁, Glu₁₂, Gln₁₄, Pro₂₀, Gly₂, Ala₁₄, Cys₂, Val₁₁, Met₂, Ile₁₂, Leu₈, Tyr₉, Phe₄, Lys₉, His₃, Trp₁, and Arg₅, with a formula molecular weight of 19,037. There is still some question about the presence of the N-terminal pyroglutamyl residue in the native protein, as cyclization may occur during isolation (Swaisgood, 1975). In addition to protein-chemical sequencing, the cDNA of -CN has been sequenced (Stewart et al., 1984), and the -CN gene sequence is complete (Alexander et al., 1988). The -CN signal peptide is composed of 21 amino acid residues, making the pre-form 190 amino acids in length.

View larger version (23K): [in this window] [in a new window]   Figure 4. Primary structure of Bos -CN A-1P (Mercier et al., 1973). The amino acid residues corresponding to the mutational differences in the B through J variants are given in Table 2. The arrow indicates the point of attack by chymosin (rennin). The * indicates pyroglutamate as the cyclized N-terminal. The site of post-translational phosphorylation (SeP) is indicated in italicized, boldface type; residues that may potentially be phosphorylated are underlined.

The bond sensitive to chymosin (EC 3.4.23.4) (rennin) hydrolysis occurs between Phe 105 and Met 106 (Figure 4) (Delfour et al., 1965; Jollés et al., 1968). The hydrolytic products are para--CN (residues 1–105) and the macropeptide (residues 106–169). Doi et al. (1979) and Vreeman et al. (1977, 1986) have observed para--CN in purified preparations of -CN. This is undoubtedly due to a chymosin-like proteolysis subsequent to translation, but more work must be done before concluding that para--CN is a natural constituent of milk or a product of storage or of the preparatory processes. It is interesting to note that of the 11 known variants, 8 occur in the distal portion of the macropeptide, relatively far removed from the point of chymosin attack. These mutations range from positions 136 to 155 and occur in the extended portion of the molecule, which serves as a physical deterrent to coagulation prior to the action of chymosin. Small, relatively neutral changes in this portion of the molecule may not adversely affect cheese making. Perhaps more interesting are the changes in the C, F², and G¹ variants, which occur in the para--CN portion of the molecule. The F² variant is functionally identical in that the net charge remains constant (Arg10His). The C variant may be of interest as Arg 97 has been implicated in the possible attraction of chymosin to the CN micelle, but the positive charge is conserved by the His 97 substitution. In a similar way, the G¹ variant in which Arg 97 is converted to Cys 97, could further influence micelle structure as the new sulfhydryl residue could promote unusual disulfide linkages close to the chymosin cleavage site.

The major component (~50%) of all -CN variants is generally believed to be the carbohydrate-free component. However, the post-translational modifications of -CN, which result in the formation of minor components, have been studied in considerable detail, and their degree of complexity is correlated with the degree of sophistication of the instrumentation used to study them. Generally, the minor -CN components are multiglycosylated and/or multiphosphorylated forms of the major -CN. Vreeman et al. (1977, 1986) concluded from their investigations that, in order of elution from DEAE-cellulose, the adsorbed -CN were the major components free of carbohydrate with one phosphate group followed by 6 minor components differing in degrees of glycosylation and phosphorylation. Doi et al. (1979) concluded from their fractionation of -CN on DEAE-cellulose that there were 4 major and 2 minor components, all containing one phosphate group and various degrees of glycosylation. The major fraction was the carbohydrate-free component.

Several researchers have investigated the structure of carbohydrate moieties (Tran and Baker, 1970; Fiat et al., 1972; Jollés et al., 1972, 1973; Fournet et al., 1975; Jollés et al., 1978). Fournet et al. (1975) isolated 3 oligosaccharides from -CN and determined the structures for 2. Saito and Itoh (1992) confirmed their structures and added 3 more; all of these structures are given in Figure 5. A composite summary of the reported glycosyl moeities, their molecular weights, and relative percentage occurrence is given in Table 3. From these data, it appears as though the complex structures C, D, and E are most prevalent. Mollé and Léonil (1995) used reverse-phase HPLC in conjunction with on-line electrospray ionization MS (EIMS) to characterize the distribution of glycosyl residues further within the -CN macropeptide for the A variant. They found at least 14 glycosylated forms, a glycosylated and non-phosphorylated form, and multiphosphorylated forms (1P at 78%, 2P at 20%, and 3P at 2%) totaling 18 reported species attributable to post-translational modification. The EIMS data on HPLC followed the absorbance data and confirmed the structures of Saito and Itoh (1992) except for A, the monosaccharide.

View larger version (14K): [in this window] [in a new window]   Figure 5. Reported structures for glycosyl residues attached to -CN macropeptide (Saito and Itoh, 1992).

View this table: [in this window] [in a new window]   Table 3. Summary of properties and occurrence of O-glycosyl residues attached to -CN macropeptides.

View this table: [in this window] [in a new window]   Table 4. Summary of the reported sites of O-glycosylation of -CN macropeptide.

From all of these data, it would appear that for -CN glycosylation, structures C, D, and E are statistically most prevalent, and Thr residues 131 and 133 are the sites most populated by these glycosyl moieties. Two reasons postulated for the prevalence of glycosyl residues at the 131 and 133 sites are that the proline turns, which bound this region, maintain its surface orientation (Kumosinski et al., 1993a) and that glycosylation at other sites is restricted by the neighboring Ile (or Val) residues (Pisano et al., 1994), which are highly prevalent in the -CN macropeptide. In studies with whole CN, it is important to remember that the major -CN bands for the A and B variants (40 to 60%) are not glycosylated; therefore, differences in the minor bands could be related to a variety of factors specific to the milk sample, not the least of which is genetic variation (Pisano et al., 1994; Mollé and Léonil, 1995).

Because of the high degree of heterogeneity and the limited amounts of the minor components of -CN, we feel that the precise nomenclature of these components still cannot be achieved at this time. We suggest that they be identified according to the genetic variant of the major nonglycosylated component and that isolated fractions, which contain post-translational modifications, be numbered consecutively according to either their increasing relative electrophoretic mobility in alkaline urea gels or their elution from anion exchange media in the presence of mercaptoethanol. For example, starting with -CN A, the nonglycosylated 1P form would be designated -1, and then subsequent bands would be termed -2, -3, -4, etc. This is in accord with most current working definitions of isolated fractions.

	THE WHEY PROTEINS

TOP ABSTRACT INTRODUCTION THE CASEINS THE WHEY PROTEINS REFERENCES

 
The term whey proteins has been used to describe the group of milk proteins that remain soluble in milk serum or whey after precipitation of CN at pH 4.6 and 20°C. Traditionally, ß-LG, -LA, serum albumin (SA), Ig, and proteose-peptone fractions have been considered the major characterized components of this fraction. Because the current and most frequently used method of assessing the integrity of this fraction is SDS-PAGE, LF, a major component that is readily visualized by this technique, should be added to this list (Figure 6). It is also recognized that proteolytic fragments of CN (Eigel et al., 1984) and fat globule membrane proteins (Mather et al., 2000) occur in the traditional whey fraction, raising questions concerning the utility of this term. However, based on current knowledge, the term whey protein should be used only in a general sense to describe milk proteins soluble at pH 4.6 and 20°C. Commercial products termed whey protein isolates or concentrates are obtained from cheese manufacture at higher pH and will contain intact caseins as well as their proteolytic products, such as macropeptides and proteose-peptone fraction. Individual families, such as ß-LG, -LA, SA, and LF, should be classified according to homology with the primary sequence of their amino acid chains. Polyacrylamide or starch gel electrophoresis still can be used to characterize and identify individual members of each family. Immunoglobulins, proteins not unique to milk, are the products of B-lymphocytes and are the result of somatic gene segment rearrangement and somatic mutation. With 1 million variants, Ig lend themselves poorly to traditional biochemical characterization. Immunochemical criteria continue to be used for laboratory diagnosis and quantitation of Ig, but molecular genetics is heavily used for structural analyses.

View larger version (109K): [in this window] [in a new window]   Figure 6. Discontinuous SDS-PAGE of dialyzed whey (pH 4.6) and individual purified whey proteins. 1 = dialyzed whey (pH 4.6); 2 = lactoferrin; 3 = BSA; 4 = bovine IgG, Cohn fraction II heavy chain (top) and light chain (bottom); 5 = ß-LG; 6 = -LA; 7 = dialyzed whey (pH 4.6); and 8 = standard purified proteins. Refer to Figure 10a for more details on Ig proteins. All samples were reduced with 2-mercaptoethanol (Basch et al., 1985).

The reference protein for this family, ß-LG B, consists of 162 amino acids and has the following composition: Asp₁₀, Asn₅, Thr₈, Ser₇, Glu₁₆, Gln₉, Pro₈, Gly₄, Ala₁₅, Cys₅, Val₉, Met₄, Ile₁₀, Leu₂₂, Tyr₄, Phe₄, Lys₁₅, His₂, Trp₂, and Arg₃. The calculated formula molecular weight is 18,277, and the measured molecular weight is 18,278.35 ± 2.2 Da (Léonil et al., 1995) or 18,277.0 ± 0.9 (Burr et al., 1997); its ExPASy entry name and file number are LACB_Bovin and P02754, respectively. The primary sequence shown in Figure 7 is unchanged since the 1984 review (Eigel et al., 1984). However, the disulfide bonds in the native protein are now unambiguously determined as Cys 66 to Cys 160 and Cys 106 to Cys 119, with Cys 121 as the source of the free thiol (Papiz et al., 1986; Bewley et al., 1997; Brittan et al., 1997; Brownlow et al., 1997; Qin et al., 1998a, b; 1999). The calculated formula weight of 18,277 takes these disulfide linkages into account.

View larger version (29K): [in this window] [in a new window]   Figure 7. Primary structure of bos ß-LG B (Eigel et al., 1984). The free sulfhydryl group is on Cys121 in the native form of the protein (Bewley et al., 1997; Brittan et al., 1997; Brownlow et al., 1997; Qin et al., 1998a,b, 1999). The two disulfide bridges are residues 66 to 160 and 106 to 120. The genetic variant substitutions are given in Table 2 for the A (Eigel et al.,1984), C (Eigel et al., 1984), D (Eigel et al., 1984), Dr. (Eigel et al., 1984), E (Eigel et al., 1984), F (Eigel et al., 1984), G (Eigel et al., 1984), I (Godovac-Zimmermann et al., 1996), J (Godovac-Zimmermann et al., 1996), and W (Godovac-Zimmermann et al., 1990) variants. The sequence positions of the major secondary structural features, -helix, helical regions, and ß-strands (ß-A to ß-I), are shown above the main sequence. The elements shown were determined for the triclinic (lattice X) form (Brownlow et al., 1997), and there are slight differences, particularly at the ends of the assigned structural elements, for the other crystal forms (Bewley et al., 1997; Qin et al., 1999) and at different pH (Qin et al., 1998a). The PDB (protein structural database) file for this protein is 1B8E.

The publication of the first high-resolution X-ray crystal structures of the triclinic form (lattice X) of ß-LG A/B (Brownlow et al., 1997), the orthorhombic form (lattice Y) of ß-LG A, B, and C (Bewley et al., 1997), and the trigonal form (lattice Z) of ß-LG A and B (Qin et al., 1998a) has verified the earlier medium resolution structure (Papiz et al., 1986) in general terms and has corrected some earlier errors. The amino acid sequences corresponding to the -helix, the ß-sheet strands (A to I), and several 3₁₀ turns for the lattice X form are indicated in Figure 7. There are slight differences among the crystal lattice forms as to the details of the secondary structural elements. In all cases, the ß-I strand is an important feature of the interface between the 2 monomers that constitute the dimer in all of the high resolution crystal structures. The NMR structures (Kuwata et al., 1999; Uhrínová et al. 2000) at about pH 2.5 contain the same strands and helix, confirming the polypeptide fold.

Another feature of bovine ß-LG is the ability to bind hydrophobic and amphiphilic molecules ranging from hexane to palmitic acid to vitamin D (Hambling et al., 1992; Pérez and Calvo, 1995; Narayan and Berliner, 1997; Sawyer, 2003). Considerable attention has been paid to the binding of retinol (vitamin A), which is essential for mammalian growth and well being, to ß-LG, and ß-LG is considered a member of the lipocalin family of proteins (Sawyer, 2003). Although some retinoids and fatty acids can bind in the deep hydrophobic pocket of ß-LG (Cho et al., 1994; Qin et al., 1998b; Wang et al., 1999; Kontopidis et al., 2002), there is some doubt about the biological role of this protein. The original biological role could have been related to maternal physiology, but this may have shifted to a more nutritional role for some species (Kontopidis et al., 2002).

The observation that ß-LG may be glycosylated (Léonil et al., 1997) prior to milking, but probably external to the mammary epithelium, is interesting and suggests a chemical rather than a biochemical reaction. More extensive modification (lactosylation) occurs in heated milk or whey (Burr et al, 1996; Léonil et al., 1997; Morgan et al., 1998), where Lys47 and Lys91 are the most reactive, and Lys8 and Lys141 are the least reactive (Morgan et al., 1998).

The practice of naming a new variant "X" until such time as the sequence has been demonstrated should save embarrassment and/or duplication of protein names. However, there is the possibility that such a ß-LG X could be confused with the X, Y, and Z lattice forms, a nomenclature used by the crystallographers. The various engineered proteins often have slight differences at the N-terminus of the protein because of the method of synthesis. (For example, Kim et al. [1997] expressed a ß-LG in which the N-terminal sequence was Glu-Ala-Glu-Ala-Tyr-Val-Thr-, whereas it is Leu-Ile-Val-Thr- in the natural proteins [Figure 7]). It is recommended that such proteins be clearly labelled so that they are not confused with the naturally synthesized proteins, because the difference in structure would give the proteins different properties, such as electrophoretic mobility.

-LA
The whey protein -LA has a specific and defined physiological function in the mammary gland. Within the Golgi apparatus of the mammary epithelial cell, -LA interacts with the ubiquitously expressed enzyme ß-1,4-galactosyltransferase to form the lactose synthase complex. -lactalbumin modifies the substrate specificity of ß-1,4-galactosyltransferase, allowing the formation of lactose from glucose and UDP-galactose. The constitutive function of ß-1,4-galactosyltransferas, to glycosylate glycoproteins and glycolipids is reversibly altered by combining with -LA in a 1:1 molar ratio. The production of lactose, its function as the major osmolyte of milk, and the function of the lactose synthase complex, have been the topics of a number of reviews (Brew and Hill, 1975; Hill and Brew, 1975; Jones, 1977; Kuhn et al., 1980).

Bovine milk contains -LA at a concentration of approximately 1.2 to 1.5 g/L (Jenness, 1974). The protein has been sequenced, and the nucleotide sequence has been confirmed (Vilotte et al., 1987; Brew et al., 1970; Bleck and Bremel, 1993a). The mature -LA (Figure 8) is a 123-amino acid globular protein (Brew et al., 1970). The -LA signal peptide is composed of 19 amino acids, making the pre-form of -LA 142 amino acids in length (Gaye et al., 1987; Hurley and Schuler, 1987). The mature -LA protein has 2 predominant genetic variants (A and B) that have been confirmed by sequence analysis (Table 2) (Bhattacharya et al., 1963). The B variant is present in the milk of most Bos taurus cattle, and both the A and B variants are found in Bos indicus cattle (Jenness, 1974). -lactalbumin A variant is present at a low frequency in some Italian and Eastern European Bos taurus breeds (Mariani and Russo, 1977). The A variant contains a Glu at position 10 of the mature protein, and the B variant has an Arg substitution at that position (Gordon, 1971). A third genetic variant, -LA C, has also been reported but not confirmed by DNA or protein sequencing (Bell et al., 1981). This variant was identified in Bali cattle (Bos javanicus). The C variant was reported to differ from the B variant by having either an Asn for Asp or a Gln for Glu substitution. The B variant is the reference protein for the family and is composed of the following amino acid residues: Ala₃, Arg₁, Asn₈, Asp₁₃, Cys₈, Gln₆, Glu₇, Gly₆, His₃, Ile₈, Leu₁₃, Lys₁₂, Met₁, Phe₄, Pro₂, Ser₇, Thr₇, Trp₄, Tyr₄, Val₆ (Brew et al., 1970). Both the A and B variant contain 4 disulfide bonds. The B variant has a formula molecular weight of 14,178; its sequence is given in Figure 8 and has been corrected since the last report to take into account the nucleic acid sequences noted previously. The ExPASy entry name for -LA is LACA_ Bovin, and its file number is P00741. -lactalbumin has a very high content of the essential amino acids (Trp, Phe, Tyr, Leu, Ile, Thr, Met, Cys, Lys, and Val). Essential amino acids account for 63.2% of the total amino acid content compared with just 51.4% for total CN (Heine et al., 1991). The amino acid composition of bovine -LA and its 72% sequence identity to human -LA makes it an ideal protein for the nutrition of human infants (Heine et al., 1991).

View larger version (17K): [in this window] [in a new window]   Figure 8. Primary sequence of Bos -LA B (Brew et al., 1970; Vanaman et al., 1970). The disulfide bridges in the molecule are between positions 6 and 120, 28 and 111, 61 and 77, and 73 and 91. The amino acid residues corresponding to the differences in genetic variants A and C are given in Table 2. The PDB (protein structural database) file for this protein is 1F6S.

In dairy cattle, the concentration of -LA in milk decreases near the end of a lactation (Caffin et al., 1985; Regester and Smithers, 1991). This is opposite of what occurs for the other major bovine milk proteins; their concentrations tend to increase as a lactation progresses (Davies and Law, 1980). The decline in -LA concentration is correlated with the decline observed in the concentration of milk lactose at the end of a lactation. Lower concentrations of -LA have also been observed in cows that have mammary infections (Caffin et al., 1985).

The protein structure, amino acid sequence, and DNA sequence of -LA are very similar to that of the c-type lysozymes (McKenzie and White, 1991), and the predicted 3-D structure of each of these 2 proteins has been analyzed in great detail (Acharya et al., 1989; McKenzie and White, 1991). Bovine -LA and bovine lysozyme show a 62.6% similarity and 35.8% identity at the amino acid level.

-Lactalbumin has been identified as a calcium metalloprotein (Hiraoka et al., 1980). The binding of calcium, zinc, and other metals to -LA and calcium’s function in the formation of native -LA have previously been reviewed (Brew and Grobler, 1992; Permyakov and Berliner, 2000). There is evidence that the binding of calcium is necessary for proper folding and disulfide bond formation of the native protein (Chrysina et al., 2000). A 2.2-Å resolution crystal structure for bovine -LA has been elucidated (Pike et al., 1996; Chrysina et al., 2000). The folding pattern for bovine -LA is distinct from the X-ray structures that were determined for baboon, human, guinea pig, and goat -LA (Acharya et al., 1989, 1990, 1991; Harata and Muraki, 1992; Pike et al., 1996; Farrell et al., 2002). This analysis has identified a distinct calcium-binding site within the native form of -LA. There is good evidence that -LA may also be a zinc-binding protein (Ren et al., 1993). In addition to its native 3-D structure, -LA can form a stable conformational state termed a molten globule under slightly denaturing conditions (Kuwajima, 1989, 1996). This state is a conformation of -LA that occurs as an intermediate step during the formation of native protein and has been studied and reviewed in great detail as a model for the process of protein folding (Kuwajima, 1989, 1996; Wijesinha-Bettoni et al., 2001; Farrell et al., 2002).

The crystal structure for lactose synthase has also been elucidated (Ramakrishnan and Qasba, 2001). The structure was created using recombinant mouse -LA and the catalytic domain of recombinant bovine ß-1,4 galactosyltransferase. The analysis indicated that the major role of -LA in the lactose synthase complex is to hold the glucose molecule and inhibit the binding of N-acetyl-glucosamine to ß-1,4 galactosyltransferase, thus allowing the attachment of glucose to UDP-galactose to form lactose.

Site-directed mutagenesis and the expression of mutant -LA in bacteria has allowed for detailed analysis of important functional -LA amino acids. Mutations made to Glu 117 or Trp 118 reduced the binding affinity of -LA for ß-1,4-galactosyltransferase, while mutations to Phe 31 or His 32 have effects on the ability of -LA to promote glucose binding to the lactose synthase complex (Grobler et al., 1994).

The genes encoding -LA and lysozyme are found on bovine chromosome 5 (Threadgill and Womack, 1990). The -LA gene is composed of 4 exons that are transcribed into a 724 base mRNA (Gaye et al., 1987; Hurley and Schuler, 1987; Vilotte et al., 1987). The coding region of -LA along with approximately 2.0 kb of 5' flanking DNA and 350 bp of 3' flanking region have been sequenced (Vilotte et al., 1987; Bleck and Bremel, 1993b). Two DNA variations have been identified in the region of the gene encoding the bovine -LA mRNA. In addition to the DNA variation that produces the A and B variants of -LA (Osta et al., 1995a), a second DNA variation has been identified in the section of the gene that encodes the 5' untranslated region of the mRNA. This polymorphism occurs at position +15 from the transcription start site (Bleck and Bremel, 1993a, b).

The importance of -LA for the normal function of the mammary gland and normal secretion of milk has been shown using mice that have the gene encoding -LA "knocked out" (Stinnakre et al., 1994; Stacey et al., 1995). Homozygous mice containing no functional copies of the -LA gene produce a milk that contains no lactose, is extremely viscous, and cannot be removed from the mammary gland by the developing pups. Hemizygous mice that contain a single copy of the functional -LA gene produce a milk that contains less lactose and has a significantly higher solids content. These studies verify the importance of -LA not only as a protein source in milk, but also as a regulator of lactose production and the secretion of milk.

SA
Serum albumin, a major protein found in blood serum, occurs in all body tissues and secretions; it has a principal role in the transport, metabolism, and distribution of ligands (Carter and Ho, 1994); it contributes to osmotic pressure of blood (Carter and Ho, 1994); and it imparts free radical protection. Bovine SA as found in milk, is physically (Polis et al., 1950) and immunologically (Coulson and Stevens, 1950) identical to blood SA. Bovine SA represents about 1.5% of total milk protein and about 8% of total whey protein. Prior to 1990, the complete amino acid sequence of BSA was thought to contain 582 amino acids and to have a calculated molecular weight of 66,267 (Brown, 1975, 1977; Reed et al., 1980). However, based on EIMS molecular weight values, Hirayama et al. (1990) observed an error in this sequence and amended the sequence to 583 amino acid residues with 17 disulfide bonds and a corrected molecular weight of 66,399. Hirayama et al. (1990) also reported a reversed amino acid sequence for the 94th and 95th positions. The amino acid composition of the latest version is Ala₄₇, Arg₂₃, Asn₁₄, Asp₄₀, Cys₃₅, Gln₂₀, Glu₅₉, Gly₁₆, His₁₇, Ile₁₄, Leu₆₁, Lys₅₉, Met₄, Phe₂₇, Pro₂₈, Ser₂₈, Thr₃₃, Trp₂, Tyr₂₀, Val₃₆. Human SA (HSA) contains 583 amino acids and has, taking the known 17 disulfide bonds into account, a formula molecular weight of 66,399. The corrected amino acid sequence for BSA is given in Figure 9; its ExPASy entry name and file number are ALBU_Bovin and P02769, respectively.

View larger version (58K): [in this window] [in a new window]   Figure 9. Normalized albumin sequence homology: bovine/human aligned sequences illustrating conserved amino acids and invariant residues. Upper line = BSA, lower line = human serum albumin. Disulfide bonds exist between Cys at aligned positions of 53 to 62, 75 to 91, 90 to 101, 123 to 168, 167 to 176, 199 to 245, 244 to 252, 264 to 278, 277 to 288, 315 to 360, 359 to 368, 391 to 437, 436 to 447, 460 to 476, 475 to 486, 513 to 558, and 557 to 566. Cys-34 is free and unbound. The PDB (protein structural database) file for human serum albumin is 1A06.

The 3-D structure of HSA has been determined by X-ray crystallography (Ho et al., 1993), and, based on sequence work, BSA could be similar. All reported SA proteins (except lamprey) are comprised of 3 homologous domains termed I, II, and III (Carter and Ho, 1994). Carter and Ho (1994) verified that each SA domain contained 2 subdomains (lA, lB, etc.) for a total of 6 subdomains. Each domain contains 10 helical regions, 2 overlapping for a total of 28 helicies, which are extensively cross-linked by 17 disulfide bonds (Eigel et al., 1984). In addition, there are 9 loops (L1 to L9) that are held together by disulfide bonds (Carter and Ho,1994). The domains are assembled to form a heart-shaped molecule within the pH range of 4.5 to 8.0 (Carter and Ho, 1994). Human SA undergoes a major reversible conformational isomerization with changes in pH, adopting a more or less compact configuration with varying conditions (Olivieri and Craievich, 1995). Measurements of the native molecular structure are roughly that of an equilateral triangle with sides of ~80 Å (each side) and depth of ~30 Å (Carter and Ho, 1994). Carter and Ho (1994) published a depiction of the molecule that shows the 3 domains, the subdomains, and the 9 loops (L1 to L9) that should be referred to when discussing dimensional aspects of this molecule. Disulfide bonds exist between Cys at positions 53 to 62, 75 to 91, 90 to 101, 123 to 168, 167 to 176, 199 to 245, 244 to 252, 264 to 278, 277 to 289, 315 to 360, 359 to 368, 391 to 437, 436 to 447, 460 to 476, 475 to 486, 513 to 558, and 557 to 566. Cys-34 is free and unbound.

Ig
Introduction and general comments.
Analysis of either whole milk or whey protein by SDS-PAGE reveals distinct bands for the Ig fraction (Figures 6 and 10a). The Ig fraction accounts for about 1% of total milk protein or about 6% of total whey protein.

View larger version (43K): [in this window] [in a new window]   Figure 10 a) Polypeptide composition of most bovine and reference Ig revealed by SDS-PAGE. Lane 1 = reference standards with molecular size indicated in kiloDaltons, lane 2 = human monoclonal IgM, lane 3 = bovine IgM, lane 4 = human monoclonal IgA, lane 5 = bovine SIgA (largest molecular weight peptide is the secretory component), lane 6 = bovine IgG2, lane 7 = bovine IgG1, lane 8 = human IgG, lane 9 = bovine light chains, and lane 10 = reference standards. Note the smaller size of the IgG2 heavy chain. Data for IgG3, IgD, and IgE are not shown. b) Model of the basic 7S Ig molecule showing 2 heavy (H) and 2 light (L) chains joined by disulfide bonds. V = variable region; C = constant region. Subscripts 1, 2, and 3 refer to the 3 C regions of the H. CHO = carbohydrate groups. Fab refers to the (top) antigen-specific portion of the Ig molecule, and Fc refers to the cell-binding effector portion of the Ig molecule (Guidry, 1985; Larson, 1992).

Although the paired V domains determine the binding specificity of the Ig molecule, the C regions determine the isotype (class) of the Ig, e.g., IgM, IgG, etc., as well as the various biological activities of Ig (Butler, 1985). Allotypes are genetic variants within the population, much like erythrocyte blood groups, and allotypic determinants are usually found in the C regions of the H and L. Idiotypes are determinants associated with the re-arranged VDJ and VJ regions of Ig (see subsequently); their expression often correlates with a particular antibody specificity.

The N-terminal V region domains, shown in Figure 10b, are actually encoded by 3 (V, D, and J) gene segments for the H and 2 (V, J) gene segments for either or L. These segments are multigenic in any one individual, e.g., humans have 95 V_H segments, 30 D_H segments, and 6 J_H segments. These segments must be somatically rearranged during B-cell development to produce one productive VDJ and one VJ rearrangement per B-cell. Because these rearranged gene segments encode the V_H and V_L domains that determine binding specificity, this multigenic arrangement can account for 10⁷ different antibodies. Added to this is the fact that sequences can vary at the junction of V to D and D to J in the H and V to J in the L ("junctional diversity"). Furthermore, these re-arrangements are the target of somatic hypermutation with a rate that is 10⁷ higher than the eucaryotic germline mutation rate. Thus, up to 10¹² different antibody specificities can be produced by one individual. Species differ in the degree to which these mechanisms are used in repertoire development, and in cattle, the pattern is unclear. This is briefly discussed subsequently and is discussed in detail elsewhere (Butler, 2004).

Each domain of an Ig, including the V regions, is folded in a common manner, known throughout biochemistry as the Ig fold. This folded structure is composed of a "ß-barrel" of antiparallel ß-pleated sheets joined at their ends by flexible polypeptide segments. In the case of the V domains, these flexible polypeptides converge at the N-terminus of the domain to form the antigen-binding site (see previous; Janeway et al., 2001).

The junctions between domains in all Ig are more susceptible to proteolytic cleavage than the ß-barrel domain structure. Thus, a wide variety of proteases attack these junctions, producing a spectrum of separate domain products. Best known are papain, trypsin, and pepsin, which attack the so-called hinge of IgG between the first and second C region domains (dark area, Figure 10b). Depending on the precise site of cleavage, a single divalent F(ab)'₂ fragment plus a 30-kDa pFc' fragment is generated (typical for pepsin), whereas, in other cases, 2 separate Fab fragments and one Fc fragment is generated (typical for papain). These fragments are useful in preparing immunochemical reagents and for identifying the location of allotypic or idiotypic determinants or testing for subclass-associated biological functions. Immunoglobulin M, IgE, and IgA are more resistant to proteolytic activity, although the latter is often attached by various "IgA proteases" (Kilian and Russell, 1998).

Another feature of Ig is that the C-terminal domain of each class (isotype) can be transcribed in 2 forms. One form contains a short membrane anchor (membrane exon); the other is transcribed with an even shorter secretion exon. The former permits any particular class of Ig to be expressed as a B-cell receptor on B lymphocyte membranes; the latter tailors Ig for secretion from the B-cell.

Interestingly, many proteins of immunological interest are encoded by genes of the Ig supergene family and include the following: a) B-cell surface receptors, b) the membrane proximal portion of the major histocompatibility complex protein, c) the T-cell receptor, d) most Fc receptors (see subsequent), e) one family of adhesion molecules, f) some cytokine receptors, and g) ß₂-microglobulin [a polypeptide chain associated with class I major histocompatibility complex molecules (Petersen et al., 1972)]. The latter was first crystallized from milk as lactollin (Groves et al., 1963) and was reviewed in the last report of this Committee (Eigel et al., 1984).

L and H Components of Bovine Ig
The extreme heterogeneity of Ig reduces the value of physical and chemical parameters for their characterization. Not surprisingly, identification of Ig has traditionally been based on immunochemical criteria rather than more conventional biochemistry. Increasingly, progress in the characterization of bovine Ig and the genes encoding them, is based on molecular genetic methods. To overcome the problem of polyclonal heterogeneity, heterohybridomas secreting bovine IgM, IgE, IgA, IgG1, and IgG2 molecules have been produced (Srikumaran, 1983, 1987; Guidry et al., 1986; Goldsby et al., 1987). Thus, homogeneous Ig proteins are now available for biochemical studies.

Although and L are nearly equally represented in humans and swine, sequence analyses by Hood et al. (1967) suggested that >90% of all bovine L are of the type. This was confirmed in the last decade, and molecular genetic methods were used to characterize many V gene segments. There are data on bovine chain sequences including those of the V regions (Ivanov et al., 1988; Sinclair et al., 1995; Parng et al., 1996). Additional information is accumulating on usage of the V region genes encoding the V_H, and V repertoire in cattle. The majority of the expressed bovine Ig H V regions are encoded by the V_H4 gene family (Saini et al., 1997; Sinclair et al., 1997), which is a departure from the usage of V_H3 in swine and rabbits. Especially noteworthy is the exceptionally long CDR3 region reported for some V regions (Saini et al., 1999). This has given rise to the idea of an additional flexible V region domain that might function in the "induced fit" concept of the antibody-binding site (Butler, 1997, 2004). Molecular genetic approaches have also allowed the genes encoding the bovine Ig C regions to be cloned, sequenced (Table 5), and their genomic organization established (Figure 11; Butler, 2004). Initial studies of this type shed light on the repertoire of genes encoding the major classes and subclasses (Knight et al., 1988; Knight and Becker, 1987), while subsequent studies provide sequence information for individual genes (Kacskovics and Butler, 1996; Brown et al., 1997; Mousavi et al., 1997; Rabbani et al., 1997; Symons et al., 1987, 1989). Zhao et al. (2002) have clarified the organization of the bovine H locus (Figure 11) and more recently characterized bovine IgD. These studies have collectively shown that only 3 IgG subclasses are functionally encoded in cattle (see Table 5 and CIgW web site).

View larger version (27K): [in this window] [in a new window]   Figure 11. Genomic organization of the heavy chain locus in cattle compared with that of human, rabbit, mouse, and camel. Highlighted area in the human depicts the duplication in the constant region locus, while the highlighted area for rabbit depicts the duplication of genes encoding IgA; here a = . The region enclosed by interrupted lines is the Sµ-C region. Note that only cattle have a S segment (Butler, 2004).

Although Ig consist of a basic 4-polypeptide chain unit of 2 H and 2 L, both IgM and IgA can form covalent polymers, pentamers, and dimers, respectively, with the addition of a 15-kDa polypeptide chain known as the J (joining) chain. The J chain plays an important role in assembly of polymeric Ig and their selective transport across epithelial cell layers. The J chain is disulfide-linked and covalently associated with the carboxyterminal tail of µ-chains and -chains (Kulseth and Rogne, 1994). Cloning and characterization of bovine Ig J chain cDNA and its promoter region have been reported (Kulseth and Rogne, 1994) and show extensive homology with the J chain from human mouse, rabbit, and bullfrog, including a highly conserved propensity to form ß-sheets. Earlier work also documented the evolutionary conservation of the J chain among higher vertebrates, as evidenced by cross-reaction of J chains from dog, cat, cow, goat, sheep, pig, horse, hedgehog, guinea pig, rat, mouse, and chicken by using rabbit antisera to human J chain (Kobayashi et al., 1973). Methods for isolation of pentameric IgM as well as its J-chain from bovine milk or colostrum have been reported (Mukkur and Froese, 1971; Komar and Mukkur, 1975; Kanno et al., 1976). The secretory component (SC) is found in milk both free and in association with IgA (Eigel et al., 1984); it is known to be a part of a polymeric Ig receptor (Kulseth et al., 1995). Its ExPasy file name is P81265.

Ig in Bovine Milk and Colostrum
Of the 5 isotypes of immunoglobulins in mammals, all have now been identified in cattle (see CIgW web site); IgG, IgA, and IgM have been characterized in milk, and their concentrations have been described and reviewed (Table 6). Changes in the level and relative proportions of the Ig in colostrum compared with milk during lactation or resulting from immunization or infections have been studied (Mackenzie and Lascelles, 1968; Butler et al., 1972; Guidry et al., 1980; Hidiroglou et al., 1992). This topic and the origin of both Ig and immunocytes in milk, especially in cattle, have been reviewed in detail (Butler, 1998; Butler and Kehrli, in press).

Immunoglobulin G1 is not the only IgG found in bovine milk and colostrum. The existence of 2 major subclasses of bovine IgG, namely IgG1 and IgG2, has been known for >3 decades, and the existence of a third subclass has been proposed (Kickhofen et al., 1968). Over the years, these have endured various changes in nomenclature, e.g., a) IgG2a, IgG2b, and IgG1 (Butler et al., 1987, 1994); b) bovine 1, 2, and 3 (Knight and Becker, 1987; Knight et al., 1988); and c) 2, 1, and Gs (Hammer et al., 1968; Kickhofen et al., 1968; Mossmann et al., 1973). The nomenclature of the Kickhofen and Mossman group is no longer used. The description of IgG2b or IgG3 was based on serological (Butler et al., 1987) and genetic studies (Knight et al., 1988), and it was speculated that the 2 are in fact identical (Heyermann et al., 1992). The work of Rabbani et al. (1997) at the Karolinska Institute has clearly confirmed the existence of 3 IgG subclasses in cattle, and, by international agreement, these are now recognized as IgG1, IgG2, and IgG3 (Table 5).

The relative proportions of IgG1 and IgG2 differ in serum and mammary secretions; whereas serum contains nearly equivalent proportions of IgG1 and IgG2, the ratio of IgG1:IgG2 in colostrum and mature milk is 15 to 20:1 and 4 to 7:1, respectively (Guidry et al., 1980). As historically shown, IgG1 is selectively transported by the mammary gland (see previous), although during inflammation (mastitis), there is substantial transudation into milk of all serum proteins including IgG2 (Mackenzie and Lascelles, 1968; Butler et al., 1972; Butler and Heyerman, 1986; Hidiroglou et al., 1992). Very little is known about the distribution and biological function of IgG3 as no IgG3-specific antibodies are currently available for such studies.

LF
A family of specific iron-binding proteins occurs in milk. The major bovine protein is LF, or lactotransferrin to distinguish it from the serum transferrin that also occurs in milk (Groves, 1960) particularly in colostrum (Groves, 1971). Lactoferrin is of mammary origin and can be found in the milk of most species (Schanbacher et al., 1993). However, LF also occurs in secretions of other epithelial cells (Masson et al., 1966), as well as in polymorphonuclear leucocytes (Baggiolini, 1972). The mammary reference protein occurs at a concentration between 20 and 200 µg/L. The actual concentration varies and increases noticeably in response to inflammation or infection. The family occurs as a single-chain polypeptide with varying degrees of glycosylation. It consists of 689 amino acids as follows: Asp₃₆, Asn₂₉, Thr₃₆, Ser₄₅, Glu₄₀, Gln₂₉, Pro₃₀, Gly₄₉, Ala₆₇, Cys₃₄, Val₄₆, Met₄, Ile₁₆, Leu₆₆, Tyr₂₁, Phe₂₇, Lys₅₄, His₁₀, Trp₁₃, Arg₃₇. Lactoferrin has a calculated molecular weight of 76,110, taking its 17 disulfide bonds into account. Measured molecular weights vary, depending primarily on the level of glycosylation (Hurley et al., 1993). The primary structure (Figure 12) was determined in the early 1990s (Mead and Tweedie, 1990; Goodman and Schanbacher, 1991; Pierce et al., 1991). The LF signal peptide is composed of 19 amino acids, making the pre-form of LF 708 amino acids in length. Lactoferrin is listed in the ExPASy database as TRFL_Bovin; its file number is P24627. The 3-D structure of bovine holo-LF at 2.8 Å determined by Moore et al. (1997) can be found in the Protein Data Bank with the identifier 1BLF (Berman et al., 2000). Lactoferrin consists of a single-polypeptide chain that folds into 2 globular lobes an N-lobe (residues 1 to 333) and a C-lobe (residues 345 to 676) connected by a 3-turn helix (residues 334 to 344). Each lobe is further divided into 2 domains (N1, residues 1 to 90 and 251 to 333, and N2, residues 91 to 250; C1, residues 345 to 431 and 593 to 676, and C2, residues 432 to 592). Bovine LF has 5 potential glycosylation sites (Figure 12) (Pierce et al., 1991), and recently Wei et al. (2000) reported glycans at each of the 5 sites. The composition of these glycans is complex and varies with the stage of lactation. At the time of its discovery, the reddish color and compositional similarity to transferrin and ovotransferrin suggested that LF might have a role in iron transport (Groves, 1960). Lactoferrin fully saturated with Fe⁺³ is called hololactoferrin and has a salmon color; in its native state, the degree of saturation is in the range of 15 to 40%. Iron-depleted LF is known as apolactoferrin. Early studies (Brown and Parry, 1974; Parry and Brown, 1974) focused on the conformational effects of iron binding as well as identification of side chain ligands. In the detailed description (Moore et al., 1997), each iron-binding site is located in a cleft between the 2 domains of a lobe. The anion, carbonate, or bicarbonate, required for iron binding, occupies a positively charged pocket in the wall of the N2 or C2 domain (Moore et al., 1997). Because the iron-LF complex is very stable, iron is released only below pH 3.5, whereas iron release by transferrin occurs in mildly acidic media of pH 5.5 (Abdallah and El Hage Chahine, 2000); it may more properly be classed as an iron scavenger than an iron transport protein.

View larger version (45K): [in this window] [in a new window]   Figure 12. Primary structure of Bos lactoferrin (LF). Disulfides occur between the following Cys: 9 and 45, 19 and 36, 115 and 198, 157 and 173, 160 and 183, 170 and 181, 231 and 245, 348 and 380, 358 and 371, 405 and 684, 425 and 647, 457 and 532, 481 and 675, 491 and 505, 502 and 515, 573 and 587, and 625 and 630. The 2 iron-binding sites include (Asp60, Tyr92, Tyr192, His253) and (Asp395, Tyr433, Tyr526, His595). The anion-binding sites are (Arg121, Thr117) and (Arg463, Thr459). The potential glycosylation sites are Asn233, Asn281, Asn368, Asn476, and Asn545. The PDB (protein structural database) file for this protein is 1BLF.

Technological Function
Because protein structure dictates function, it should not be surprising that a protein with the complex structure of LF would prove to be multifunctional. Also, not surprising is the attractiveness of such a multifunctional protein for commercial development. Despite the low concentration of LF in bovine milk, it is now being isolated and purified on an industrial scale from cheese whey, mainly in Japan and Europe (Horton, 1995). Among its useful characteristics, the heat stability of LF is such that typical pasteurization processes have little effect on structure (Sanchez et al., 1992). Development is beginning on commercial applications utilizing bovine LF and its partially digested peptides as neutriceuticals in infant formulas, health supplements, oral care products, and animal feeds to capitalize on its ability to boost natural defense against infections (Sanchez et al., 1992). Its potential for use as an antioxidant is beginning to see realization in cosmetics (Steinjns and van Hooijdonk, 2000). The commercial production of LF has given impetus to research on lactoferricin(s), 15-residue peptides in pepsin hydrolysates of LF (Strom et al., 2000), which have antimicrobial activity of their own (Strom et al., 2002).

Over the past decade, LF has become that rare protein for which there is comprehensive structural data and a multitude of functions. In recognition of this, 5 biennial conferences dealing solely with lactoferrin have now been held, most recently in 2001 (Conference Abstracts, 2002).

View larger version (58K): [in this window] [in a new window]   Figure 9 (Continued). Normalized albumin sequence homology: bovine/human aligned sequences illustrating conserved amino acids and invariant residues. Upper line = BSA, lower line = human serum albumin. Disulfide bonds exist between Cys at aligned positions of 53 to 62, 75 to 91, 90 to 101, 123 to 168, 167 to 176, 199 to 245, 244 to 252, 264 to 278, 277 to 288, 315 to 360, 359 to 368, 391 to 437, 436 to 447, 460 to 476, 475 to 486, 513 to 558, and 557 to 566. Cys-34 is free and unbound. The PDB (protein structural database) file for human serum albumin is 1A06.

	REFERENCES

TOP ABSTRACT INTRODUCTION THE CASEINS THE WHEY PROTEINS REFERENCES

 


Aalund, G. D., J. E. Butler, J. R. Duncan, M. J. Freeman, R. Jenness, J. M. Kehoe, J.-P. Mach, J. Rapacz, M.-P. Vaerman, and A. J. Winter. 1971. Proposed nomenclature for the immunoglobulins of the domesticated Bovidae. Can. J. Compo. Med. 35:346–348.

Abdallah, F. B., and J. M. El Hage Chahine. 2000. Transferrins: Iron release from lactoferrin. J. Mol. Biol. 303:255–266.[Medline]

Acharya, K. R., J. S. Ren, D. I. Stuart, R. E. Fenna, and D. C. Phillips. 1991. Crystal structureof human -lactalbumin at 1.7 Å resolution. J. Mol. Biol. 221:571–581.[Medline]

Acharya, K. R., D. I. Stuart, D. C. Phillips, and H. A. Scheraga. 1990. A critical evaluation of the predicted and X-ray structures of -lactalbumin. J. Prot. Chem. 9:549–563.[Medline]

Acharya, K. R., D. I. Stuart, N. P. C. Walker, M. Lewis, and D. C. Phillips. 1989. Refined structure of baboon -lactalbumin at 1.7 Å resolution. Comparison with c-type lysozyme. J. Mol. Biol. 208:99–127.[Medline]

Aleandri, R., L. G. Buttazzoni, J. C. Schneider, A. Caroli, and R. Davoli. 1990. The effects of milk protein polymorphisms on milk components and cheese-producing ability. J. Dairy Sci. 73:241–255.[Abstract]

Alexander, L. J., G. Hayes, W. Bawden, A. F. Stewart, and A. G. Mackinlay. 1993. Complete nucleotide sequence of bovine ß-lactoglobulin gene. Anim. Biotechnol. 4:1–10.

Alexander, L. J., A. F. Stewart, A. G. Mackinlay, T. V. Kapelinskaya, T. M. Tkach, and S. I. Gorodetsky. 1988. Isolation and characterization of the bovine -casein gene. Eur. J. Biochem. 178:395–401.[Medline]

Ambrosius, H., R. Asofsky, R. A. Binaghi, D. Capra, L. Hood, E. Orlans, D. S. Rowe, P. Small, G. Torrigiani, and J.-P. Vaerman. 1978. Proposed rules for the designation of immunoglobulins of animal origin. Bull. World Health Org. 56:815–817.[Medline]

Aoki, T., K. Toyooka, and Y. Kako. 1985. Role of phosphate groups in the calcium sensitivity of _S2-casein. J. Dairy Sci. 68:1624–1629.[Abstract/Free Full Text]

Aoki, T., N. Yamada, and Y. Kako. 1990. Relation between colloidal calcium phosphate cross-linkage and release of ß-casein from bovine casein micelles on cooling. Agric. Biol. Chem. 54:2287–2292.

Arnold, R. R., M. F. Cole, and A. McGhee, Jr. 1977. A bactericidal effect for human lactoferrin. Science 197:263–265.[Abstract/Free Full Text]

Aschaffenburg, R., and J. Drewry. 1957. Genetics of the ß-lactoglobulins of cow’s milk. Nature (Lond.) 180:376–378.

Baggiolini, M. 1972. The enzymes of the granules of polymorphonuclear leukocytes and their functions. Enzyme 13:132–60.[Medline]

Baranyi, M., Z. Bösze, J. Buchberger, and I. Krause. 1993. Genetic polymorphism of milk proteins in Hungarian Spotted and Hungarian Grey cattle: A possible new genetic variant of ß-lactoglobulin. J. Dairy Sci. 76:630–636.[Abstract/Free Full Text]

Barman, T. E. 1970. Purification and properties of bovine milk glyco--lactalbumin. Biochem. Biophys. Acta 214:242–244.[Medline]

Basch, J. J., F. W. Douglass, Jr., L. G. Prochino, V. H. Holsinger, and H. M. Farrell, Jr. 1985. Quantitation of caseins and whey proteins of processed milks and whey protein concentrates, application of gel electrophoresis, and comparison with Harland-Ashworth procedure. J. Dairy Sci. 68:23–31.[Abstract/Free Full Text]

Bech, A. M., and K. R. Kristiansen. 1990. Milk protein polymorphism in Danish dairy cattle and the influence of genetic variants on milk yield. J. Dairy Res. 57:53–62.[Medline]

Beeby, R. 1964. The presence of sulphydryl groups in k-casein. Biochim. Biophys. Acta 82:418–419.[Medline]

Beh, K. J. 1973. Distribution of Brucella antibody among immunoglobulin classes and a low molecular weight antibody fraction in serum and whey of cattle. Res. Vet. Sci. 14:381–384.[Medline]

Bell, K., K. E. Hopper, and H. A. McKenzie. 1981. Bovine -lactalbumin C and s1-, ß-, and -caseins of Bali (Banteng) cattle, Bos (Bibos) javanicus. Aust. J. Biol. Sci. 34:149–159.[Medline]

Berman, H. M., J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, and P. E. Bourne. 2000. The protein data bank. Nucleic Acids Res. 28:235–242.[Abstract/Free Full Text]

Bewley, M. C., B. Y. Qin, G. B. Jameson, L. Sawyer, and E. N. Baker. 1997. ß-Lactoglobulin and its variants: A three-dimensional structural perspective. Pages 100–109 in Milk Protein Polymorphism. Int. Dairy Fed. Special Issue 9702. Int. Dairy Fed., Brussels, Belgium.

Bhattacharya, S. D., A. K. Roychoudhury, N. K. Sinha, and A. Sen. 1963. Inherited -lactalbumin and ß-lactoglobulin polymorphism in Indian Zebu cattle. Comparison of Zebu and buffalo -lactalbumins. Nature 197:797–799.[Medline]

Bigelow, C. C. 1967. On the average hydrophobicity of proteins and the relation between it and protein structure. J. Theoret. Biol. 16:187–211.[Medline]

Bingham, E. W., N. Parris, and H. M. Farrell, Jr. 1988. Phosphorylation of ß-casein and -lactalbumin by casein kinase from lactating bovine mammary gland. J. Dairy Sci. 71:324–336.

Blakeslee, D., J. E. Butler, and W. H. Stone. 1971. Serum antigens of cattle. II. Immunogenetics of two immunoglobulin allotypes. J. Immunol. 107:227–235.[Abstract/Free Full Text]

Bleck, G. T., and R. D. Bremel. 1993a. Correlation of the -lactalbumin (+15) polymorphism to milk production and milk composition of Holsteins. J. Dairy Sci. 76:2292–2298.[Abstract]

Bleck, G. T., and R. D. Bremel. 1993b. Sequence and single-base polymorphisms of the bovine -lactalbumin 5' flanking region. Gene 126:213–218.[Medline]

Bokhout, B. A. 1975a. Divergent results in radial immunodiffusion with antisera differing in precipitation properties with respect to individual immunoglobulin subclasses. I. The antigen factor. J. Immunol. Methods 7:187–198.[Medline]

Bokhout, B. A. 1975b. Divergent results in radial immunodiffusion with antisera differing in precipitation properties with respect to individual immunoglobulin subclasses. II. The antiserum factor. J. Immunol. Methods 7:199–210.[Medline]

Bokhout, B. A., and J. J. vanAsten-Noordijk. 1979. Divergent results in radial immunodiffusion. III. Isolation and specificity test of class-specific antibodies. J. Immunol. Methods 27:101–109.[Medline]

Bonsing, J., J. M. Ring, A. F. Stewart, and A. G. Mackinlay. 1988. Complete neucleotide sequence of the bovine ß-casein gene. Aust. J. Biol. Sci. 41:527–537.[Medline]

Bouniol, C., C. Printz, J.-C. Mercier. 1993. Bovine _S2-casein D is generated by exon VIII skipping. Gene 128:289–293.[Medline]

Brandon, M. R., D. L. Watson, and A. K. Lascelles. 1971. The mechanism of transfer of immunoglobulin into mammary secretion of cows. Aust. J. Exp. Biol. Med. Sci. 49:613–623.[Medline]

Brew, K., F. J. Castellino, T. C. Vanaman, and R. L. Hill. 1970. The complete amino acid sequence of bovine -lactalbumin. J. Biol. Chem. 245:4570–4582.[Abstract/Free Full Text]

Brew, K., and J. Grobler. 1992. -Lactalbumin. Pages 191–229 in Advanced Dairy Chemistry, Proteins. 2nd ed. P. F. Fox, ed. Elsevier Sci. Pub. Ltd., New York, NY.

Brew, K., and. R. L. Hill. 1975. Lactose biosynthesis. Rev. Physiol. Biochem. Pharmacol. 72:105–158.[Medline]

Brignon, G., B. Ribadeau Dumas, J.-C. Mercier, J.-P. Pelissier, and B. C. Das. 1977. The complete amino acid sequence of bovine _S2 -casein. FEBS Lett. 76:274–279.[Medline]

Brittan, H., J. C. Mudford, G. E. Norris, T. M. Kitson, and J. P. Hill. 1997. Labeling the free sulfydryl group in ß-lactoglobulin A, B and C. Pages 200–203 in Milk Protein Polymorphism. Int. Dairy Fed. Special Issue 9702. Int. Dairy Fed., Brussels, Belgium.

Brown, J. R. 1975. Structure of bovine serum albumin. Fed. Proc. FASEB 34:591.

Brown, J. R. 1977. Structure and evolution of serum albumin. Pages 27–51 in Albumin Structure, Function, and Uses. V. M. Rosenoer, M. Oratz, and M. A. Rothschild, ed. Pergamon Press, Oxford, UK.

Brown, E. M., and R. M. Parry, Jr. 1974. A spectroscopic study of bovine lactoferrin. Biochemistry 13:4560–4565.[Medline]

Brown, W. R., H. Rabbani, J. E. Butler, and L. Hammarström. 1997. Characterization of a bovine C alpha gene. Immunology (British) 91:1–6.

Brownlow, S., J. H. Morais-Cabral, R. Cooper, D. R. Flower, S. J. Yewdall, I. Polikarpov, A. C. T. North, and L. Sawyer. 1997. Bovine ß-lactoglobulin at 1.8 Å resolution—Still an enigmatic lipocalin. Structure 5:481–495.[Medline]

Bullen, J. J., H. J. Rogers, and E. Griffiths. 1978. Role of iron in bacterial infection. Curr. Top. Microbiol. Immunol. 80:1–35.[Medline]

Burr, R., C. H. Moore, and J. P. Hill. 1996. Evidence of multiple glycosylation of bovine ß-lactoglobulin by electrospray ionization mass spectrometry. Milchwissenschaft. 51:488–492.

Burr, R. G., C. H. Moore, and J. P. Hill. 1997. ESI-MS phenotyping of bovine ß-lactoglobulin genetic variants in a New Zealand dairy cattle population. Pages 340–344 in Milk Protein Polymorphism. Int. Dairy Fed. Special Issue 9702. Int. Dairy Fed., Brussels, Belgium.

Butler, J. E. 1969. Bovine immunoglobulins: A review. J. Dairy Sci. 52:1895–1909.[Abstract/Free Full Text]

Butler, J. E. 1973. The occurrence of immunoglobulin fragments, two types of lactoferrin and a lactoferrin-IgG2 complex in bovine colostral and milk whey. Biochim. Biophys. Acta 295:341–351.[Medline]

Butler, J. E. 1974. Immunoglobulins of the mammary secretions. Pages 217–255 in Lactation, Vol. III. B. L. Larson and V. R. Smith, ed. Academic Press, New York, NY.

Butler, J. E. 1983. Bovine immunoglobulins: An augmented review. Vet. Immunol. Immunopathol. 4:43–151.[Medline]

Butler, J. E. 1985. Biochemistry and biology of ruminant immunoglobulins. Pages 1–53 in Progress in Veterinary Microbiology and Immunology. Vol. 2. R. Pandey, ed. S. Karger, Basel, Switzerland.

Butler, J. E. 1997. Immunoglobulin gene organization and the mechanism of repertoire development. Scand. J. Immunol. 45:455–462.[Medline]

Butler, J. E. 1998. Immunoglobulins and immune cells in animal milks. Pages 1531–1554 in Mucosal Immunology. P. L. Ogra, J. Mestecky, M. E. Lamm, W. Strober, J. R. McGhee, and J. Bienenstock, ed. Academic Press, New York, NY.

Butler, J. E. 2004. Diparate mechanisms drive antibody diversity among animals. Trends Immunol. (accepted).

Butler, J. E., and H. Heyermann. 1986. The heterogeneity of bovine IgG2 - I. The Al allotypic determinant is the major antigenic determinant recognized on bovine IgG2a by polyclonal rabbit anti-IgG2a. Mol. Immunol. 23:291–296.[Medline]

Butler, J. E., H. Heyermann, L. V. Freyno, and J. Kiernan. 1987. The heterogeneity of bovine IgG2. II. The identification of IgG2b. Immunol. Lett. 16:31–38.[Medline]

Butler, J. E., and M. E. Kehrli, Jr. 2004. Immunocytes and immunoglobulins in milk. In Mucosal Immunology, 3rd ed. P. L. Ogra, J. Mestecky, M. E. Lamm, W. Strober, J. R. McGhee, and J. Bienenstock, ed. Academic Press, New York, NY (accepted).

Butler, J. E., C. F. Maxwell, C. S. Pierce, M. B. Hylton, R. Asofsky, and C. A. Kiddy. 1972. Studies on the relative synthesis and distribution of IgA and IgGi in various tissues and body fluids of the cow. J. Immunol. 109:38–46.[Abstract/Free Full Text]

Butler, J. E., P. Navarro, and H. Heyermann. 1994. Heterogeneity of bovine IgG2. VI. Comparative specificity of monoclonal and polyclonal capture antibodies for IgG2a (A2) and IoG2a (A9). Vet. Immunol. Immunopathol. 40:119–133.[Medline]

Caffin, J. P., B. Poutrel, and P. Rainard. 1985. Physiological and pathological factors influencing bovine -lactalbumin and ß-Iactoglobulin concentrations in milk. J. Dairy Sci. 68:1087–1094.

Carles, C., J.-C. Huet, and B. Ribadeau-Dumus. 1988. KA new strategy for primary structure determination of proteins: Application to ß-casein. FEBS Lett. 229:265–272.[Medline]

Carter, D. C., and J. X. Ho. 1994. Structure of serum albumin. Adv. Protein Chem. 45:153–203.[Medline]

Cho, Y., C. A. Batt, and L. Sawyer. 1994. Probing the retinol-binding site of bovine ß-lactoglobulin. J. Biol. Chem. 269:11102–11107.[Abstract/Free Full Text]

Choi, B. K., and R. Jimenez-Flores. 1996. Study of putative glycosylation sites in bovine ß-casein introduced by PCR-based site-directed mutagenesis. J. Agric. Food Chem. 44:358–364.

Chrysina, E. D., K. Brew, and K. R. Acharya. 2000. Crystal structures of apo- and holo-bovine -lactalbumin at 2.2- Å resolution reveal an effect of calcium on inter-lobe interactions. J. Biol. Chem. 275:37021–37029.[Abstract/Free Full Text]

Chung, E. R., S. K. Han, and T. J. Rhim. 1995. Milk protein polymorphisms as genetic markers in Korean native cattle. Asian-Aust. J. Anim. Sci. 8:187–194.

Conference Abstracts. 2002. Abstracts of 5th Int. Conf. Lactoferrin: Structure, Function and Applications. Biochem. Cell Biol. 80:137–168.

Conti, A., L. Napolitano, A. M. Cantisani, R. Davoli, and S. Dall’Olio. 1988. Bovine ß-lactoglobulin H: Isolation by preparative isoelectric focusing in immobilized pH gradients and preliminary characterization. J. Biochem. Biophys. Methods 16:205–214.[Medline]

Corradini, C. 1969. Distribution of the genetic variants _s1-, ß-, and -caseins in milk from Jersey cows in The Netherlands. Neth. Milk Dairy J. 23:79–82.

Coulson, E. J., and H. Stevens. 1950. The serological relationship of bovine whey albumin to serum albumin. J. Biol. Chem. 187:355–363.[Free Full Text]

Creamer, L. K., H. F. Zoerb, N. F. Olson, and T. Richardson. 1982. Surface hydrophobicity of _s1-I, _s1- casein A and B and its implications in cheese structure. J. Dairy Sci. 65:902–906.[Abstract/Free Full Text]

Davies, D. T., and A. J. R. Law. 1980. The content and composition of protein in creamery milks in south-west Scotland. J. Dairy Res. 47:83–90.

Davoli, R., S. Dall’Olio, and D. Bigi. 1988. A new ß-lactoglobulin variant in bovine milk. Scienza-e-Tecnica-Lattiero-Casearia 39:439–443.

Delfour, A., J. Jolles, C. Alais, and P. Jolles. 1965. Caseino-glycopeptides: Characterization of a methionine residue and of the N–terminal sequence. Biochem. Biophys. Res. Comm. 19:452–455.[Medline]

Dixon, F. J., W. O. Weigle, and J. J. Vazques. 1961. Metabolism and mammary secretion of serum proteins in the cow. Lab. Invest. 10:216–237.[Medline]

Doi, H., F. Ibuki, and M. Kanamori. 1979. Heterogeneity of reduced bovine -casein. J. Dairy Sci. 62:195–203.[Abstract/Free Full Text]

Doi, H., H. Kobatake, F. Ibuki, and M. Kanamori. 1980. Attachment sites of carbohydrate portions to peptide chain of -casein from bovine colostrum. Agric. Biol. Chem. 44:2605–2611.

Dong, C., and K. F. Ng-Kwai-Hang. 1998. Characterization of a non-electrophoretic genetic variant of ß-casein by peptide mapping and mass spectroscopic analysis. Int. Dairy J. 8:967–972.

Duncan, J. R., B. N. Wilkie, F. Hiestand, and A. J. Winter. 1972. The serum and secretory immunoglobulins of cattle: Characterization and quantitation. J. Immunol. 108:965–976.[Abstract/Free Full Text]

Eigel, W. N., J. E. Butler, C. A. Ernstrom, H. M. Farrell, Jr., V. R. Harwalkar, R. Jenness, and R. M. Whitney. 1984. Nomenclature of proteins of cow’s milk: Fifth revision. J. Dairy Sci. 67:1599–1631.[Abstract/Free Full Text]

Erhardt, G. 1989. -Kasine in rindermilch-nachweis weiterer allels (ß-CN E) in verschiedener rassen. J. Anim. Breed. Gen. 106:225–231.

Erhardt, G. 1993. A new _s1-casein allele in bovine milk and its occurrence in different breeds. Anim. Genet. 24:65–66.[Medline]

Erhardt, G. 1996. Detection of a new -casein variant in the milk of pinzgauer cattle. Anim. Gen. 27:105–107.

Farrell, H. M., Jr., T. F. Kumosinski, P. H. Cooke, G. King, P. D. Hoagland, E. D. Wickham, H. J. Dower, and M. L. Groves. 1996. Particle sizes of purified -casein: Metal effect and correspondence with predicted three-dimensional structures. J. Prot. Chem. 15:435–445.[Medline]

Farrell, H. M., Jr., T. F. Kumonsinski, P. Pulaski, M. P. Thompson. 1988. Calcium-induced associations of the caseins: A thermodynamic linkage approach to precipitation and resolubilization. Arch. Biochem. Biophys. 265:146–158.[Medline]

Farrell, H. M., Jr., P. X. Qi, E. M. Brown, P. H. Cooke, M. H. Tunick, E. D. Wickham, and J. J. Unruh. 2002. Molten globule structures in milk proteins: Implications for potential new structure-function relationships. J. Dairy Sci. 85:459–471.[Abstract]

Fiat, A.-M., C. Alais, and P. Jolles. 1972. The amino-acid and carbohydrate sequences of a short glycopeptide isolated from bovine -casein. Eur. J. Biochem. 27:408–412.[Medline]

Formaggioni, P., A. Summer, M. Maracarne, and P. Mariani. 1999. Milk protein polymorphism: Detection and diffusion of the genetic variants in Bos genus. Annal. della Fac. di Med. Vet. XIX:127–165.

Fournet, B., A.-M Fiat, C. Alais, and P. Jollés. 1979. Cow -casein: Structure of the carbohydrate portion. Biochim. Biophys. Acta 576:339–346.[Medline]

Fournet, B., A.-M. Fiat, J. Montreuil, and P. Jollés. 1975. The sugar part of -caseins from cow milk and colostrum and its microheterogeneity. Biochimie 57:161–165.[Medline]

Gaye, P., D. Hue-Delahaie, J.-C. Mercier, S. Soulier, J.-L. Vilotte, and J.-P. Furet. 1987. Complete nucleotide sequence of ovine -lactalbumin mRNA. Biochimie 69:601–608.[Medline]

Geldermann, H., J. Gogol, M. Kock, and G. Tacea. 1996. DNA variants within the 5' flanking region of bovine milk protein encoding genes. J. Anim. Breed. Gen. 113(4–5):261–267.

Godger, B. V. 1971. A low molecular weight protein in bovine serum with similar electrophoretic mobility to IgG₂. Res. Vet. Sci. 12:465–468.[Medline]

Godovac-Zimmermann, J., I. Krause, M. Baranyi, S. Fischer-Fruhholz, J. Juszczak, G. Erhardt, J. Buchberger, and H. Klostermeyer. 1996. Isolation and rapid sequence characterization of two novel bovine ß-lactoglobulins I and J. J. Protein Chem. 15:743–750.[Medline]

Godovac-Zimmermann, J., I. Krause, J. Buchberger, G. Weiss, and H. Klostermeyer. 1990. Genetic variants of bovine ß-lactoglobulin. A novel wild-type ß-Lactoglobulin W and its primary sequence. Biol. Chem. Hoppe-Seyler 371:255–260.

Goldsby, R. A., S. Srikumaran, A. Arulanandanl, B. Hague, F. A. Ponce de Leon, M. Sevoian, and A. J. Guidry. 1987. The application of hybridoma technology to the study of bovine immunoglobulins. Vet. Immunol. Immunopathol. 17:25–35.[Medline]

Goodman, R. E., and F. L. Schanbacher. 1991. Bovine lactoferrin mRNA: Sequence, analysis, and expression in the mammary gland. Biochem. Biophys. Res. Comm. 180:75–84.[Medline]

Gorczyca, W., M. Ugorski, W. Nowacki, and J. Lisowski J. 1986. Immunoglobulins of colostrum. – VI. Comparative studies of cytophilic properties of bovine serum and colostral IgG. Mol. Immunol. 23:961–964.[Medline]

Gordon, W. G. 1971. -Lactalbumin. Pages 331–363 in Milk Proteins: Vol. II, H. A. McKenzie, ed. Academic Press, New York, NY.

Gouldsworthy, A. M., J. Leaver, and J. M. Banks. 1996. Application of a mass spectrometry sequencing technique for identifying peptides present in cheddar cheese. Int. Dairy J. 6:781–791.

Grobler, J. A., M. Wang, A. C. W. Pike, and K. Brew. 1994. Study by mutagenesis of the roles of two aromatic clusters of -lactalbumin in aspects of its action in the lactose synthase system. J. Biol. Chem. 269:5106–5114.[Abstract/Free Full Text]

Groenen, M. A. M., R. E. M. Dijkhof, A. J. M. Verstege, and J. J. van der Poel. 1993. The complete sequence of the gene encoding bovine _S2-casein. Gene 123:187–193.[Medline]

Grosclaude, F. 1988. Le polymorphisme genetique des principales 1actoproteines bovines. Relations avec la quantité, la composition et les aptitudes fromageres du lait. INRA Prod. Anim. 1:5–17.

Grosclaude, F., P. Joudrier, and M. F. Mahé. 1978. Polymorphisme de la caseine _S2 bovine: Etroite liaison du locus _S2-Cn avec les loci _s1-Cn, ß-Cn et -Cn; mise en evidence d’une deletion dans le variant _S2-Cn D. Ann. Genet. Sel. Anim. 10:313–327.

Grosclaude, F., M. F. Mahé, and J. P. Accolas. 1982. Note sur le polymorphisme genetique des lactoproteines de bovins et de yaks Mongols. Ann. Genet. Sel. Anim. 14:545–550.

Grosclaude, F., M. F. Mahé, and J.-C. Mercier, 1974. Comparaison du polymorphisme genetique des Lactoproteins du Zebu et des bovines. Ann Génet. Sél Anim. 6:305–329.

Grosclaude, F., M. F. Mahé, J.-C. Mercier, J. Bonnemaire, and J. H. Teissier. 1976. Polymorphisme des lactoproteines de bovines Nepalais. Polymorphisme des caseine _s2-mineurs. Ann. Genet. Sel. Evol. 8:461–479.

Grosclaude, F., M. F. Mahé, J.-C. Mercier, and B. Ribadeau-Dumas. 1970. The localization of a deletion of 13 amino acids differentiating variant A from variants B and C of bovine _s1-casein in the –terminal part. FEBS Lett. 11:109–112.[Medline]

Grosclaude, F., M. F. Mahé, and B. Ribadeau-Dumas. 1973. Structure primaire de la caseine _s1-et de la caseine ß-bovine. Eur. J. Biochem. 40:323–324.[Medline]

Groves, M. L. 1960. The isolation of a red protein from milk. J. Am. Chem. Soc. 82:3345–3350.

Groves, M. L. 1969. Some minor components of casein and other phosphoproteins in milk. A review. J. Dairy Sci. 52:1155–1165.[Abstract/Free Full Text]

Groves, M. L. 1971. Minor milk proteins and enzymes. Pages 367–418 in Milk Proteins, Vol. II. H. A. McKenzie, ed. Academic Press, New York, NY.

Groves, M. L., J. J Basch, and W. G. Gordon. 1963. Isolation, characterization and amino acid composition of a new crystalline protein, lactollin from milk. Biochemistry 2:2814–817.

Groves, M. L., H. J. Dower, and H. M. Farrell, Jr. 1992. Reexamination of the polymeric distributions of -casein isolated from bovine milk. J. Prot. Chem. 11:21–28.[Medline]

Groves, M. L., E. D. Wickham, and H. M. Farrell, Jr. 1998. Environmental effects on disulfide bonding patterns of bovine in -casein. J. Prot. Chem. 17:73–84.[Medline]

Guidry, A. J. 1985. Mastitis and the immune system of the mammary gland. Pages 229–262 in Lactation. B. L. Larson, ed. Iowa State University Press, Ames.

Guidry, A. J., J. E. Butler, R. B. Pearson, and B. T. Weinland. 1980. IgA, IgG₁, IgG₂, IgM, and BSA in serum and mammary secretion throughout lactation. Vet. Immunol. Immunopathol. 1:329–341.

Guidry, A. J., S. Srikumaran, and R. A. Goldsby. 1986. Production and characterization of bovine immunoglobulins from bovine X murine hybridomas. Methods Enzymol. 121:244–265.[Medline]

Hambling S. G., A. S. McAlpine, and L. Sawyer. 1992. ß-Lactoglobulin. Pages 141–190 in Advanced Dairy Chemistry, Vol. 1. Proteins. P. F. Fox, ed. Elsevier Applied Science, London, United Kingdom.

Hammer, D. K., B. Kickhofen, and G. Henning. 1968. Molecular classes and properties of antibodies in cattle serum and colostrum synthesized during the primary and secondary response to protein antigens. Eur. J. Biochem. 6:443–454.[Medline]

Han, S. K., Y. C. Shin, and H. D. Byun. 2000. Biochemical, molecular and physiological characterization of a new ß-casein variant detected in Korean cattle. Anim. Gent. 31:49–51.

Harata, K., and M. Muraki. 1992. X-ray structural evidence for a local helix-loop transition in -lactalbumin. J. Biol. Chem. 267:1419–1421.[Abstract/Free Full Text]

Heine, W. E., P. D. Klein, and P. J. Reeds. 1991. The importance of -lactalbumin in infant nutrition. J. Nutr. 121:277–283.

Heyermann, H., and J. E. Butler. 1987. The heterogeneity of bovine IgG₂. IV. Structural differences between IgG_2a molecules of the Al and A2 allotypes. Mol. Immunol. 24:1327–1334.[Medline]

Heyermann, H., J. E. Butler, and B. Frangione. 1992. The heterogeneity of bovine IgG₂. V. Differences in the primary structure of bovine IgG₂ allotypes. Mol. Immunol. 29:1147–1152.[Medline]

Hidiroglou, M., T. R. Batra, and K. H. Nielsen. 1992. Effect of vitamin E supplementation and of health status of mammary gland on immunoglobulin concentration in milk of dairy cows. Ann. Rech. Vet. 23:139–144.[Medline]

Hill, J. P., M. J. Boland, L. K. Creamer, S. G. Anema, D. E. Otter, G. R. Paterson, R. Lowe, R. L. Motion, and W. C. Thresher. 1996. Effect of bovine ß-Iactoglobulin phenotype on the properties of ß-lactoglobulin, milk composition, and dairy products. Pages 281–294 in Macromolecular Interactions in Food Technology. ACS Symp. Ser. 650. N. Parris, A. Kato, L. K. Creamer, and R. J. Pearce, ed. Amer. Chem. Soc., Washington, DC.

Hill, R. L., and K. Brew. 1975. Lactose synthetase. Adv. Enzymol. Relat. Areas Mol. Biol. 43:411–490.[Medline]

Hiraoka, Y., T. Segawa, K. Kuwajima, S. Sugai, and N. Murai. 1980. -Lactalbumin: A calcium metalloprotein. Biochem. Biophys. Res. Comm. 95:1098–1104.[Medline]

Hirayama, K., S. Akashi, M. Furuya, and K. Fukuhara. 1990. Rapid confirmation and revision of the primary structure of bovine serum albumin by ESIMS and Frit-FAB LC/MS. Biochem. Biophys. Res. Commun. 173:639–646.[Medline]

Ho, J. X., E. W. Holowachuk, E. J. Norton, P. D. Twigg, and D. C. Carter. 1993. X-ray and primary structure of horse serum albumin (Equus caballus) at 0.27-nm resolution. Eur. J. Biochem. 215:205–212.[Medline]

Hoagland, P. D., J. J. Unruh, E. D. Wickham, and H. M. Farrell, Jr. 2001. Secondary structure of bovine _S2-casein: Theoretical and experimental approaches. J. Dairy Sci. 84:1944–1949.[Abstract]

Hood, L., W. R. Gray, B. G. Sanders, and W. J. Dreyer. 1967. Light chain evolution. Cold Spring Harbor Symp. Quant. Biol. 32:133–146.[Abstract/Free Full Text]

Horton, B. S. 1995. Commercial utilization of minor milk components in the health and food industries. J. Dairy Sci. 78:2584–2589.[Abstract]

Hurley, W. L., R. C. J. Grieve, C. E. Magura. H. M. Hegarty, and S. Zou. 1993. Electrophoretic comparisons of lactoferrin from bovine mammary secretions, milk neutrophils, and human milk. J. Dairy Sci. 76:377–387.[Abstract/Free Full Text]

Hurley, W. L., and L. A. Schuler. 1987. Molecular cloning and nucleotide sequence of a bovine -lactalbumin cDNA. Gene 61:119–123.[Medline]

Ivanov, V. N., V. A. Karginov, I. V. Morozov, and S. I. Gorodetsky. 1988. Molecular cloning of a bovine immunoglobulin lambda chain cDNA. Gene 67:41–48.[Medline]

Jakob, E., and Z. Puhan. 1992. Technological properties of milk as influenced by genetic polymorphism of milk proteins—A review. Int. Dairy J. 2:157–178.

Janeway, C. A., P. Travers, M. Walport, and M. Shlomchik. 2001. Immunology, 5th ed. Garland Publishing, New York, NY.

Jann, O., G. Ceriotti, A. Caroli, and G. Erhardt. 2002. A new variant in exon VII of the bovine ß-casein gene (CSN2) and its distribution among European cattle breeds. J. Anim. Breed. Genet. 119:65–68.

Jenness, R. 1974. The composition of milk. Pages 3–107 in Lactation, Vol. III. B. L. Larson and V. R. Smith, ed. Academic Press, New York, NY.

Jenness, R., B. L. Larson, T. L. McMeekin, A. M. Swanson, C. H. Whitnah, and R. M. Whitney. 1956. Nomenclature of the proteins of bovine milk. J. Dairy Sci. 39:536–541.[Abstract/Free Full Text]

Jimenez-Flores, R., Y. C. Kang, and T. Richardson. 1987. Cloning and sequence analysis of bovine ß-casein cDNA. Biochem. Biophys. Res. Comm. 142:617–621.[Medline]

Jollés, J., C. Alais, and P. Jollés. 1968. The tryptic peptide with the rennin-sensitive linkage of cow’s -casein. Biochim. Biophys. Acta 168:591–593.[Medline]

Jollés, J., A.-M. Fiat, C. Alais, and P. Jollés. 1973. Comparative study of cow and sheep -caseino-glycopeptides: Determination of the N–tenninal sequences with a sequencer and the location of the sugars. FEBS Lett. 30:173–176.[Medline]

Jollés, P., M. H. Loucheux-Lefebvre, and A. Henschen. 1978. Structural relatedness of k-casein and fibrinogen -chain. J. Mol. Evol. 11:271–277.[Medline]

Jollés, J., F. Schoentgen, C. Alais, A.-M. Fiat, and P. Jollés. 1972. Studies on the primary structure of cow k-casein. Structural features of para--casein: N-terminal sequence of -caseino-glycopeptide studied with a sequencer. Helv. Chim. Acta 55:2872–2883.[Medline]

Jones, E. A. 1977. Synthesis and secretion of milk sugars. Symp. Zool. Soc. London 41:77–92.

Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottsman, and C. Foeller. 1991. In Sequences of Proteins of Immunological Interest. 5th ed. NIH Publ. No. 91–3242. US Dept. Health Human Serv., US Govt. Printing Office, Washington, DC.

Kacskovics, I., and J. E. Butler. 1996. The heterogeneity of bovine IgG2. VIII. The complete cDNA sequence of bovine IgG2a(A2) and an IgG1. Mol. Immunol. 33:189–195.[Medline]

Kacskovics, I., T. E. Wittum, J. E. Butler, and E. T. Littledike. 1995. The heterogeneity of bovine IgG2. VII. The phenotypic distribution of the Al and A2 allotypes of IgG2a among beef cows with known clinical history. Vet. Immunol. Immunopathol. 48:89–96.[Medline]

Kacskovics, I., Z. Wu, N. Simister, V. L. Frenyo, and L. Hammarström. 2000. Cloning and characterization of bovine MHC class I-like Fc receptor. J. Immunol. 164:1889–1897.[Abstract/Free Full Text]

Kaminogawa, S., K. Yamauchi, and C.-H. Yoon. 1980. Calcium insensitivity and other properties of _S1-I casein. J. Dairy Sci. 63:223–227.[Abstract/Free Full Text]

Kanamori, M., N. Kawaguchi, F. Ibuki, and H. Doi. 1980. Attachment sites of carbohydrate. moieties to peptide chain of bovine k-casein from normal milk. Agric. Biol. Chem. 44:1855–1861.

Kanno, C., D. B. Emmons, V. R. Harwalkar, and J. A. Elliott. 1976. Purification and characterization of the agglutinating factor for lactic streptococci from bovine milk: IgM-immunoglobulin. J. Dairy Sci. 59:2036–2045.[Abstract/Free Full Text]

Kemler, R., H. Mossmann, U. Strohmaier, B. Kickhöfen, and D. K. Hammer. 1975. In vitro studies on the selective binding of IgG from different species to tissue sections of the bovine mammary gland. Eur. J. Immunol. 5:603–608.[Medline]

Kickhofen, B., D. K. Hammer, and D. Scheel. 1968. Isolation and characterization of G type immunoglobulins from bovine serum and colostrum. Hoppe-Seyler’s Z. Physiol. Chem. 349:1755–1773.[Medline]

Kilian, M., and M. W. Russell. 1998. Microbial evasion of IgA functions. Pages 241–251 in Mucosal Immunology. P. L. Ogra, J. Mestecky, M. E. Lamm, W. Strober, J. Bienenstock, and J. R. McGhee, ed. Academic Press, New York, NY.

Kim, T.-R., Y. Goto, N. Hirota, K. Kuwata, H. Denton, S.-Y. Wu, L. Sawyer, and C. A. Batt. 1997. High-level expression of bovine ß-lactoglobulin in Pichia pastoris and characterization of its physical properties. Prot. Eng. 10:1339–1345.[Abstract/Free Full Text]

Knight, K. L., and R. S. Becker. 1987. Isolation of genes encoding bovine IgM, IgG, IgA and IgB chains. Vet. Immunol. Immunopathol. 7:17–24.

Knight, K. L., M. Suter, and R. S. Becker. 1988. Genetic engineering of bovine Ig. Construction and characterization of hapten-binding bovine/murine chimeric IgE, IgA, IgG₁, IgG₂ and IgG₃ molecules. J. Immunol. 140:3654–3659.[Abstract]

Kobayashi, K., J.-P. Vaerman, H. Bazin, A.-M. Lebacq-Verheyden, and J. F. Heremans. 1973. Identification of J-chain in polymeric immunoglobulins from a variety of species by cross-reaction with rabbit antisera to human J-chain. J. Immunol. 111:1590–1594.[Abstract/Free Full Text]

Koczan, D., G. Hobom, and H.-M. Seyfert. 1991. Genomic organization of the bovine _S1-casein gene. Nucleic Acids Res. 19:5591–5596.[Abstract/Free Full Text]

Komar, R., and T. K. S. Mukkur. 1975. Isolation and characterization of J-chain from bovine colostral immunoglobulin M. Can. J. Biochem. 53:943–949.[Medline]

Kontopidis, G., C. Holt, and L. Sawyer. 2002. The ligand-binding site of bovine ß-lactoglobulin: Evidence for a function? J. Mol. Bol. 318:1043–1055.

Kuhn, N. J., D. T. Carrick, and C. J. Wilde. 1980. Lactose synthesis: Possibilities of regulation. J. Dairy Sci. 63:328–336.

Kulczycki, A. J., Jr., G. S. Nash, M. J. Bertovich, H. D. Burack, and R. P. MacDermott. 1987. Bovine milk IgG, but not serum IgG, inhibits pokeweed mitogen-induced antibody secretion by human peripheral blood mononuclear cell. J. Clin. Immunol. 7:37–45.[Medline]

Kulseth, M. A., and S. Rogne. 1994. Cloning and characterization of the bovine immunoglobulin J. chain cDNA and its promoter region. DNA Cell Biol. 13:37–42.[Medline]

Kulseth, M. A., P. Krajci, O. Myklebost, and S. Rogne. 1995. Cloning and characteristication of two forms of bovine polymeric immumoglobulin receptor DNA. DNA Cell Biol. 14:251–256.[Medline]

Kumosinski, T. F., E. M. Brown, and H. M. Farrell, Jr. 1993a. Three dimensional molecular modeling of bovine caseins: A refined energy-minimized -casein structure. J. Dairy Sci. 76:2507–2520.[Abstract]

Kumosinski, T. F., E. M. Brown, and H. M. Farrell, Jr. 1993b. Three-dimensional molecular modeling of bovine caseins: an energy-minimized ß-casein structure. J. Dairy Sci. 76:931–945.[Abstract/Free Full Text]

Kumosinski, T. F., G. King, and H. M. Farrell, Jr. 1994. An energy minimized three dimensional working model for casein submicelles. J. Prot. Chem. 13:681–700.[Medline]

Kuwajima, K. 1989. The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins: Struct. Func. Genet. 6:87–103.

Kuwajima, K. 1996. The molten globule state of a-lactalbumin. FASEB J. 10:102–109.[Abstract]

Kuwata, K., M. Hoshino, V. Forge, S. Era, C. A. Batt, and Y. Goto. 1999. Solution structure and dynamics of bovine ß-lactoglobulin A. Protein Sci. 8:2541–2545.[Medline]

Larson, B. L. 1992. Immunoglobulins of the mammary secretions. Pages 231–254 in Advanced Dairy Chemistry-I: Proteins. P. F. Fox, ed. Elsevier Applied Science, New York, NY.

Le Bars, D., and J.-C. Grippon 1989. Specificity of plasmin towards bovine _S2-casein. J. Dairy Res. 56:817–821.[Medline]

Le Franc, M.-P., and G. Le Franc. 2001. The Immunoglobulin Facts Book. Academic Press, San Diego, CA.

Léonil, J., D. Mollé, J. Fauquant, J. L. Maubois, R. J. Pearce, and S. Bouhallab. 1997. Characterization by ionization mass spectrometry of lactosyl ß-lactoglobulin conjugates formed during heat treatment of milk and whey and identification of one lactose-binding site. J. Dairy Sci. 80:2270–2281.[Abstract]

Léonil, J., D. Mollé, F. Gaucheron, P. Arpin, P. Guenot, and J. L. Maubois. 1995. Analysis of major bovine milk proteins by on-line high-performance liquid chromatography and electro spray ionization-mass spectrometry. Lait 75:193–210.

Lisowski, J., M. Janusz, B. Tyran, A. Morawiecki, S. Golab, and H. Bialkowska. 1975. Immunoglobulins of colostrum -IV. Comparative studies of bovine serum and colostral IgG1 and IgG2. Immunochemistry 12:167–172.

Mackenzie, D. D. S., and A. K. Lascelles. 1968. The transfer of I-131-labeled immunoglobulins and serum albumin from blood into milk of lactating ewes. Aust. J. Exp. Biol. Med. Sci. 46:285–294.[Medline]

Mackinlay, A. G., R. J. Hill, and R. G. Wake. 1966. The action of rennin on -casein. The heterogeneity and origin of the insoluble products. Biochim. Biophys. Acta 115:103–112.[Medline]

Mackinlay, A. G., and R. G. Wake. 1965. Fractionation of S-carboxymethyl--casein and characterization of the components. Biochim. Biophys. Acta 104:167–180.[Medline]

Mahé, M. F., and F. Grosclaude. 1982. Polymorphisme de la caseine _S2 des bovines: Characterization du variant C du yak (Bos grunniens). Ann. Genet. Sel. Anim. 14:401–416.

Mahé, M. F., G. Miranda, R. Queral, A. Bado, P. Souvenir-Zafidrajaona, and F. Grosclaude. 1999. Genetic polymorphism of milk proteins in African Bos taurus and Bos indicus populations characterization of variants _S1-Cn H and -Cn J. Génét. Sél. Evol. 31:239–253.

Mariani, P., and V. Russo. 1975. Varianti genetiche delle proteine del latte nella razza Rendena. Riv. Zootec. Vet. 3:345–348.

Mariani, P., and V. Russo. 1977. Polmorfismo genetico della -lattalbumina nelle razze bovine. Riv. Zootec. Vet. 6:603–610.

Mariani, P., A. Summer, A. Anghinetti, C. Senese, P. Di Gregorio, P. Rando, and P. Serventi. 1995. Effects of the _sl-CN G allele on the percentage distribution of caseins _s1-, _S2-, ß-, and - in Italian Brown cows. Ind. Latte 31:3–13.

Masson P. L., J. F. Heremans, and C. Dive 1966. An iron-binding protein common to many external secretions. Clin. Chim. Acta 14:735–738.

Mather, I. H. 2000. A review and proposed nomenclature for major proteins of the milk-fat globule membrane. J. Dairy Sci. 83:203–247.[Abstract]

Mayans, M.O., W. J. Coadwell, D. Beale, D. B. A. Symons, and S. J. Perkins. 1995. Demonstration by pulsed neutron scattering that the arrangement of the Fab and Fc fragments in the overall structures of bovine IgG1 and IgG2 in solution is similar. Biochem. J. 311:283–291.

Mayer, B., A. Zolnai, L. V. Frenyo, V. Jancsik, Z. Szentirmay, L. Hammarström, and I. Kacskovics. 2002. Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs. Immunology 107:288–296.[Medline]

McKenzie, H. A., and F. H. White. 1991. Lysozyme and -lactalbumin: structure, function, and interrelations. Adv. Prot. Chem. 41:173–315.[Medline]

McKnight, R. A., R. Jimenez-Flores, Y. Kang, L. K. Creamer, and T. Richardson. 1989. Cloning and sequencing of a complimentary deoxyribonucleic acid coding for a bovine _s1-casein A from mammary tissue of a homozygous B variant cow. J. Dairy Sci. 72:2464–2473.

McSweeney, P. L. H., N. F. Olson, P. F. Fox, and A. Healy. 1994. Proteolysis of bovine _S2-casein by chy-mosin. Z. Lebensm. Unters. Forsch. 119:429–432.

Mead, P. E., and J. W. Tweedie. 1990. cDNA and protein sequence of bovine lactoferrin. Nucleic Acids Res. 18:7167.[Free Full Text]

Mercier, J.-C. 1981. Phosphorylation of the caseins, present evidence for an amino acid triplet code posttranslationally recognized by specific kinases. Biochimie 63:1–17.[Medline]

Mercier, J.-C., G. Brignon, and B. Ribadeau-Dumas. 1973. Structure primarie de la caseine -bovine B. Sequence complete. Eur. J. Biochem. 35:222–235.[Medline]

Mercier, J.-C., F. Grosclaude, and B. Ribadeau-Dumas. 1971. Structure primaire de la caseine _s1 bovine. Sequence complete. Eur. J. Biochem. 23:41–51.[Medline]

Miranda, G., P. Anglade, M. F. Mahe, and G. Erhardt. 1993. Biochemical characterization of the bovin genetic kappa-casein C and E variants. Anim. Genet. 4:27–31.

Mohr, U., D. Koczan, D. Linder, G. Hobom, and G. Erhardt. 1994. A single point mutation results in A allele-specific exon skipping in the bovine _s1-casein mRNA. Gene 143:187–192.[Medline]

Mollé, D., and J. Léonil. 1995. Heterogeneity of the bovine -casein caseino-macropeptide, resolved by liquid chromatography on-line with electrospray ionization mass spectrometry. J. Chromatogr. A 708:223–230.[Medline]

Moore, S. A, B. F. Anderson, C. R. Groom, M. Haridas, and E. N. Baker. 1997. Three-dimensional structure of diferric bovine lactoferrin at 2.8 A resolution. J. Mol. Biol. 274:222–236.[Medline]

Morgan, F., S. Bouhallab, D. Mollé, G. Henry, J.-L. Maubois, and J. Léonil. 1998. Lactolation of ß-lactoglobulin monitored by electrospray ionisation mass spectrometry. Int. Dairy J. 8:95–98.

Morgan, K. L., F. J. Bourne, T. J. Newby, and P. A. Bradley. 1981. Humoral factors in the secretory immune system of ruminants. Pages 391–411 in The Ruminant Immune System. J. E. Butler, ed. Plenum Press, New York, NY.

Mossmann, H., K. Bartsch, B. Rude, B. Kickhofen, and D. K. Hammer. 1973. The influence of the net charge of antigen on the distribution of bovine IgG class antibodies. Eur. J. Immunol. 3:293–298.[Medline]

Mousavi, M., H. H. Rabbani, and L. Hammarström. 1997. Characterization of the bovine epsilon gene. Immunology 92:369–373.[Medline]

Mukkur, T. K. S., and A. Froese. 1971. Isolation and characterization of IgM from bovine colostral whey. Immunochemistry 8:257–264.[Medline]

Mulvihill, D. M., and P. F. Fox. 1979. Proteolytic specificity of chymosin on bovine _S1-I casein. J. Dairy Res. 46:641–651.

Nagao, M., M. Maki, R. Sasaki, and H. Chiba. 1984. Isolation and sequence analysis of bovine _s1-casein cDNA clone. Agric. Biol. Chem. 48:1663–1667.

Narayan, M., and L. J. Berliner. 1997. Fatty acids and retinoids bind independently and simultaneously to ß-lactoglobulin. Biochemistry 36:1906–1911.[Medline]

Neelin, J. M. 1964. Variants of -casein revealed by improved starch gel electrophoresis. J. Dairy Sci. 47:506–509.[Abstract/Free Full Text]

Ng-Kwai-Hang, K. F., and F. Grosclaude. 1992. Genetic polymorphism of milk proteins. Pages 405–455 in Advanced Dairy Chemistry-1 Proteins. P. F. Fox, ed. Elsevier Applied Science, New York, NY.

Ng-Kwai-Hang, K. F., and F. Grosclaude. 2003. Genetic polymorphism of milk proteins. Pages 739–816 in Advanced Dairy Chemistry, Vol. 1, part B, Proteins. 3rd ed. P. F. Fox and P. L. H. McSweeney, ed. Kluwer Academic/Plenum, New York, NY.

Ng-Kwai-Hang, K. F., J. F. Hayes, J. E. Moxley, and H. G. Monardes. 1984. Association of genetic variants of casein and milk serum proteins with milk, fat, and protein production by dairy cattle. J. Dairy Sci. 67:835–840.

Ng-Kwai-Hang, K. F., J. F. Hayes, J. E. Moxley, and H. G. Monardes. 1986. Relationships between milk protein polymorphisms and major milk constituents in Hoistein-Friesian cows. J. Dairy Sci. 69:22–26.[Abstract/Free Full Text]

Olivieri, J. R., and A. F. Craievich. 1995. The subdomain structure of human serum albumin in solution under different pH conditions studied by small angle x-ray scattering. Eur. Biophys. J. 24:77–84.[Medline]

Osta, R., E. Garcia-Muro, and I. Zarazaga. 1995a. A MspI polymorphism at the bovine -lactalbumin gene. Anim. Gen. 26:204–205.

Osta, R., S. Marcos, and C. Rodellar. 1995b. A MnlI polymorphism at the bovine _s2-casein gene. Anim. Genet. 26:213.[Medline]

Papiz, M. Z., L. Sawyer, E. E. Eliopoulis, A. C. T. North, J. B. C. Findlay, R. Sivaprasadarao, T. A. Jones, M. E. Newcomer, and P. J. Kraulis. 1986. The structure of ß-lactoglobulin and its similarity to plasma retinol binding protein. Nature 324:383–385.[Medline]

Parng, C. L., S. Hansal, R. A. Goldsby, and B. A. Osborne. 1996. Gene conversion contributes to immunoglobulin light chain diversification in cattle. J. Immunol. 157:5478–5486.[Abstract]

Parry, R. M., Jr., and E. M. Brown 1974. Lactoferrin conformation and metal binding properties. Pages 141–160 in Advances in Experimental Medicine and Biology: Protein-Metal Interactions. M. Friedman, ed. Plenum Press, New York, NY.

Pérez, M. D., and M. Calvo. 1995. Interaction of ß-lactoglobulin with retinol and fatty acids and its role as a possible biological function for this protein: A review. J. Dairy Sci. 78:978–988.[Abstract]

Permyakov, E. A., and L. J. Berliner. 2000. -Lactalbumin: Structure and function. FEBS Lett. 473:269–274.[Medline]

Peterson, P. A., B. A. Cunningham, I. Berggard, and G. M. Edelman. 1972. ß₂-Microglobulin—A free immunoglobulin domain. Proc. Natl. Acad. Sci. USA 69:1697–1701.[Abstract/Free Full Text]

Pierce, A., D. Colavizza, M. Benaissa, P. Maes, A. Tartar, J. Montreuil, and G. Spik. 1991. Molecular cloning and sequence analysis of bovine lactoferrin. Eur. J. Biochem. 196:177–184.[Medline]

Pike, A. C. W., K. Brew, and K. R. Acharya. 1996. Crystal structures of guinea-pig, goat, and bovine -lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in lactose synthase. Structure 4:691–703.[Medline]

Pisano, A., N. H. Packer, J. W. Redmond, K. L. Williams, and A. A. Gooley. 1994. Characterization of O-linked glycosylation motifs in the glycopeptide domain of bovine -casein. Glycobiology 4:837–844.[Abstract/Free Full Text]

Pless, D. D., and W. J. Lennarz. 1977. Enzymatic conversion of proteins to glycoproteins. Proc. Natl. Acad. Sci. USA 74:134–138.[Abstract/Free Full Text]

Polis, B. D., H. W. Shmukler, and J. H. Custer. 1950. Isolation of a crystalline albumin from milk. J. Biol. Chem. 187:349–354.[Free Full Text]

Prinzenberg, E.-M., S. Hiendleder, T. Ikonen, and G. Erhardt. 1996. Molecular genetic characterization of new bovine -casein alleles CSN3-F and CSN3-G and genotyping by PCR.-RFLP. Anim. Gen. 27:347–349.

Prinzenberg, E. M., I. Krause, and G. Erhardt. 1999. SCCP analysis of the bovine CSN3 locus discriminates six alleles corresponding to known protein variants (A, B, C, E, F, G) and three new DNA polymorphism (H, I, A¹). Anim. Biotechnol. 10:49–62.[Medline]

Pujolle, J., B. Ribadeau-Dumas, J. Garnier, and R. Pion. 1966. A study of -casein components. I. Preparation. Evidence for a common C-terminal sequence. Biochem. Biophys. Res. Comm. 25:285–290.[Medline]

Qin, B. Y., M. C. Bewley, L. K. Creamer, E. N. Baker, and G. B. Jameson. 1999. Functional implications of structural differences between variants A and B of bovine ß-lactoglobulin. Protein Sci. 8:75–83.[Medline]

Qin, B. Y., M. C. Bewley, L. K. Creamer, H. M. Baker, E. N. Baker, and G. B. Jameson. 1998a. Structural basis of the Tanford transition of bovine ß-lactoglobulin. Biochemistry 37:14014–14023.[Medline]

Qin, B. Y., L. K. Creamer, E. N. Baker, and G. B. Jameson. 1998b. 12-Bromododecanoic acid binds inside the calyx of bovine ß-lactoglobulin. FEBS Lett. 438:272–278.[Medline]

Rabbani, H., W. R. Brown, J. E. Butler, and L. Hammarström. 1997. Polymorphism of the IgHG3 gene in cattle. Immunogenetics 46:326–331.[Medline]

Ramakrishnan, B., and P. K. Qasba. 2001. Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the ß-1,4-galactosyltransferase-I. J. Mol. Biol. 310:205–218.[Medline]

Rapacz, J., and J. Hasler-Rapacz. 1972. _l-Globulin allotype CI in cattle. Anim. Blood Biochem. Genet. 3 (Suppl. 1):26–27.

Rasmussen, L. K., P. Hojrup, and T. E. Petersen. 1992. Localization of two interchain disulfide bridges in dimers of bovine _S2-casein. Eur. J. Biochem. 203:381–386.[Medline]

Rasmussen, L. K., P. Hojrup, and T. E. Petersen. 1994. Disulphide arrangement in bovine caseins: Localization of intrachain disulphide bridges in monomers of - and _S2-casein from bovine milk. J. Dairy Res. 61:485–493.[Medline]

Reddy, M. I., and A. W. Mahoney. 1991. Binding of Fe(lII) to bovine _s1-casein. J. Dairy Sci. 74 (Suppl 1):D58. (Abstr.)

Reed, R. G., F. W. Putnam, and T. Peters, Jr. 1980. Sequence of residues 400–403 of bovine serum albumin. Biochem. J. 191:867–868.[Medline]

Regester, G. O., and G. W. Smithers. 1991. Seasonal changes in the ß-Iactoglobulin, -lactalbumin, glycomacropeptide, and casein content of whey protein concentrate. J. Dairy Sci. 74:796–802.[Abstract]

Ren, J., D. I. Stuart, and K. R. Acharya. 1993. -Lactalbumin possesses a distinct zinc binding site. J. Biol. Chem. 268:19292–19298.[Abstract/Free Full Text]

Ribadeau-Dumas, B., G. Brignon, F. Grosclaude, and J.-C. Mercier. 1972. Structure primaire de la casein ß bovine. Eur. J. Biochem. 25:505–514.[Medline]

Sadler, A. M., C. A. Kiddy, R. E. McCann, and W. A. Mattingly. 1968. Acid production and curd toughness in milks of different _s1-casein types. J. Dairy Sci. 51:28–35.[Abstract/Free Full Text]

Saini, S. S., B. Allore, R. M. Jacobs, and A. Kaushik. 1999. Exceptionally long CDR3H region with multiple cysteine resides in functional bovine IgM antibodies. Eur. J. Immunol. 29:2420–2426.[Medline]

Saini, S. S., W. R. Hein, and A. Kaushik. 1997. A single predominantly expressed polymorphic immunoglobulin V_H gene family related to mammalian group I, clan II, is identified in cattle. Mol. Immunol. 34:641–651.[Medline]

Saito, T., and T. Itoh. 1992. Variation and distributions of O-glycosidically linked sugar chains in bovine -casein. J. Dairy Sci. 75:1768–1774.[Abstract]

Sanchez, L., J. M. Peiro, H. Castillo, M. D. Perez, J. M. Ena, and M. Calvo. 1992. Kinetic parameters for denaturation of bovine milk lactoferrin. J. Food Sci. 57:873–879.

Sang, B. C., B. S. Ahn, B. D. Sang, Y. Y. Cho, and A. Djajanegara. 1994. Association of genetic variants of milk proteins with lactation traits in Holstein cows. Pages 217–211 in 7th AAAP Anim. Sci. Congr. Proc.

Sawyer, L. 2003. ß-Lactoglobulin. Pages 319–386 in Advanced Dairy Chemistry, Vol. 1 Proteins. 3rd ed. part A. P. F. Fox and P. L. H. McSweeney, ed. Kluwer Academic/Plenum Publishers, New York, NY.

Schanbacher, F. O., R. E. Goodman, and R. S. Talhouk. 1993. Bovine mammary lactoferrin: Implications from messenger ribonucleic acid (mRNA) sequence and regulation contrary to other milk proteins. J. Dairy Sci. 76:3812–3831.[Abstract/Free Full Text]

Schlee, P., and O. Rottman. 1992. Identification of bovine -casein C using the polymerase chain reaction. J. Anim. Breed. Genet. 109:153–155.

Schmidt, D. G. 1970. Differences between the association of the genetic variants B, C and D of _s1-casein. Biochim. Biophys. Acta 221:140–142.[Medline]

Senocq, D., D. Mollé, S. Pochet, J. Léonil, D. Dupont, and D. Levieux. 2002. A new bovine ß-casein genetic variant characterized by a Met93 Leu93 substitution in the sequence A². Lait 82:171–180.

Shahani, K. M., W. J. Harper, R. G. Jensen, R. M. Parry, Jr., and C. A. Zittle. 1973. Enzymes in bovine milk: A review. J. Dairy Sci. 56:531–543.

Sinclair, M. C., J. Gilchrist, and R. A. Aitken. 1995. Molecular characterization of bovine V lambda regions. J. Immunol. 155:3068–3078.[Abstract]

Sinclair, M. C., J. Gilchrist, and R. Aitken. 1997. The bovine IgG repertoire is dominated by a single diversified V_H gene family. J. Immunol. 159:3883–3889.[Abstract]

Singh, H., A. Flynn, and P. F. Fox. 1989. Binding of zinc to bovine and human milk proteins. J. Dairy Res. 56:235–248.[Medline]

Smith K. L., and F. L. Schanbacher. 1977. Lactoferrin as a factor of resistance to infection of the bovine mammary gland. JAVMA 170:1224–1227.

Snoeren, T. H. M., B. van Markwijk, and R. van Montfort. 1980. Some physicochemical properties of bovine _s2-casein. Biochim. Biophys. Acta 622:268–276.[Medline]

Srikumaran, S., R. A. Goldsby, A. J. Guidry, B. Hague, D. V. Onisk, and P. Srikumaran. 1987. Library of monoclonal bovine immunoglobulins and monoclonal antibodies to bovine immunoglobulins. Hybridoma 6:527–530.[Medline]

Srikumaran, S., A. J. Guidry, and R. A. Goldsby. 1983. Production and characterization of monoclonal bovine immunoglobulins G 1, G2 and M from bovine x murine hybridomas. Vet. Immunol. Immunopathol. 5:323–342.

Stacey, A., A. Schnieke, M. Kerr, A. Scott, C. McKee, I. Cottingham, B. Binas, C. Widle, and A. Colman. 1995. Lactation is disrupted by -lactalbumin deficiency and can be restored by human -lactalbumin gene replacement in mice. Proc. Natl. Acad. Sci. USA 92:2835–2839.[Abstract/Free Full Text]

Steijns, J. M, and A. C. M. van Hooijdonk. 2000. Occurrence, structure, biochemical properties and technological characteristics of lactoferrin. Br. J. Nutr. 84:S11–S17.

Stewart, A. F., J. Bonsing, C. W. Beattie, F. Shah, I. M. Willis, and A. G. Mackinlay. 1987. Complete nucleotide sequences of bovine _s2- and ß-casein cDNAs: Comparisons with related sequences in other species. Mol. Biol. Evol. 4:231–241.[Abstract]

Stewart, A. F., I. M. Wills, and A. G. Mackinlay. 1984. Neucleotide sequence of bovine _s1- and -casein cDNA’s. Nucleic Acid Res. 12:3895–3907.[Abstract/Free Full Text]

Stinnakre, M. G., J. L. Vilotte, S. Soulier, and J.-C. Mercier. 1994. Creation and phenotypic analysis of -lactalbumin-deficient mice. Proc. Natl. Acad. Sci. USA 91:6544–6548.[Abstract/Free Full Text]

Strom, M. B., B. E. Haug, O. Rekdal, M. L. Skar, W. Stensen, and J. S. Svendsen. 2002. Important structural features of IS-residue lactoferrin derivatives and methods for improvement of antimicrobial activity. Biochem. Cell Biol. 80:65–74.[Medline]

Strom, M. B., O. Rekdal, and J. S. Svendsen. 2000. Antibacterial activity of 15-residue lactoferrin derviatives. J. Peptide Res. 56:265–274.[Medline]

Sulimova, G. E., I. N. Badagueva, and I. G. Udina. 1996. Polymorphism of the -casein gene in subfamilies of the Bovidae. Genetika (Moskva) 32:1576–1582.

Sulimova, G. E., S. S. Sokolova, O. P. Semikozova, L. M. Nguet, and E. M. Berberov. 1992. Analysis of DNA polymorphisms of clustered genes in cattle: casein genes and genes of the major histocompatibility complex (BOLA). Tsitol. I Genetika 26:18–26.

Swaisgood, H. E. 1973. The caseins. CRC Crit. Rev. Food Technol. 3:375–414.

Swaisgood, H. E. 1975. Primary sequence of kappa-casein. J. Dairy Sci. 58:583–592.

Swaisgood, H. E. 1982. Chemistry of milk proteins. Pages 1–59 in Developments in Dairy Chemistry-1: Proteins. P. F. Fox, ed. Applied Science Publishers, New York, NY.

Swaisgood, H. E. 1992. Chemistry of the caseins. Pages 63–110 in Advanced Dairy Chemistry-I: Proteins. P. F. Fox, ed. Elsevier Applied Science, New York, NY.

Swaisgood, H. E. 1995. Enzymes indigenous to bovine milk. Pages 472–475 in Handbook of Milk Composition. R. G. Jensen, ed. Academic Press, New York, NY.

Swaisgood, H. E., and J. R. Brunner. 1963. Characteristics of -casein in the presence of various dissociating agents. Biochem. Biophys. Res. Comm. 12:148–151.[Medline]

Swaisgood, H. E., J. R. Brunner, and H. A. Lillevik. 1964. Physical parameters of k-casein from cow’s milk. Biochemistry 3:1616–1623.

Swaisgood, H. E., B. L. Larson, E. B. Kalan, J. R. Brunner, C. V. Morr, and P. M. T. Hansen. 1975. Methods of Gel Electrophoresis of Milk Proteins. Am. Dairy Sci. Assoc., Champaign, IL.

Symons, D. B. A., C. A. Clarkson, and D. Beale. 1989. Structure of bovine immunoglobulin constant region heavy chain gamma 1 and gamma 2 genes. Mol. Immunol. 26:841–850.[Medline]

Symons, D. B. A., C. A. Clarkson, C. P. Milstein, N. R. Brown, and D. Beale. 1987. DNA sequence analysis of two bovine immunoglobulin CR gamma pseudogenes. J. Immunogenet. 14:273–283.[Medline]

Tewari, U. J., and T. K. Mukkur. 1975. Isolation and physico-chemical characterization of bovine serum and colostral immunoglobulin G (IgG) subclasses. Immunochemistry 12:925–930.[Medline]

Thompson, M. P., and H. M. Farrell, Jr. 1974. Genetic variants of the milk proteins. Pages 109–134 in Lactation, vol. III. B. L. Larson and V. R. Smith, ed. Academic Press, New York, NY.

Threadgill, D. W., and J. E. Womack. 1990. Genomic analysis of the major bovine milk protein genes. Nucleic Acid Res. 18:6935–6942.[Abstract/Free Full Text]

Toma, S. J., and S. Nakai. 1973. Calcium sensitivity and molecular weight of _S2-casein. J. Dairy Sci. 56:1559–1562.

Tran, V. D., and B. E. Baker. 1970. Casein. IX. Carbohydrate moiety of k-casein. J. Dairy Sci. 53:1009–1021.

Uhrínová, S., M. H. Smith, G. B. Jameson, D. Uhrín, L. Sawyer, and P. N. Barlow. 2000. Structural changes accompanying pH-induced dissociation of the ß-lactoglobulin dimer. Biochemistry 39:3565–3574.[Medline]

Van der Strate, B. W. A., L. Beljaars, G. Molema, M. C. Harmsen, and D. K. F. Meijer. 2001. Antiviral activities of lactoferrin. (Review) Antiviral Res. 52:225–239.[Medline]

Vanaman, T. C., K. Brew, and R. L. Hill. 1970. The disulfide bonds of bovine -lactalbumin. J. Biol. Chem. 245:4583–4590.[Abstract/Free Full Text]

Vilotte, J. L., S. Soulier, J.-C. Mercier, P. Gaye, D. Hue-Delahaie, and J. P. Furet. 1987. Complete nucleotide sequence of bovine -lactalbumin gene: Comparison with its rat counterpart. Biochimie 69:609–620.[Medline]

Visser, S., C. J. Slangen, A. C. Alting, and H. J. Vreeman. 1989. Specificity of bovine plasmin in its action on bovine _S2-casein. Milchwissenschaft 44:335–339.

Visser, S., C. J. Slangen, F. M. Lagerwerf, W. D. Vandongen, and J. Haverkamp. 1995. Identification of a new genetic variant of bovine ß-casein using reversed-phase high-performance liquid chromatography and mass spectrometric analysis. J. Chromat. A 711:141–150.[Medline]

Vogel, H. J., D. J. Schibli, W. Jing, E. M. Lohmeier-Vogel, R. F. Epand, and R. M. Epand. 2002. Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides. Biochem. Cell Biol. 80:49–63.[Medline]

Vreeman, H. J., P. Both, J. A. Brinkhuis, and C. Van der Spek. 1977. Purification and some physicochemical properties of bovine -casein. Biochim. Biophys. Acta 491:93–103.[Medline]

Vreeman, H. J., and J. A. M. van Riel. 1990. The large-scale isolation of _S2-casein from bovine casein. Neth Milk Dairy J. 44:43–48.

Vreeman, H. J., S. Visser, C. J. Slangen, and J. A. M. van Riel. 1986. Characterization of bovine -casein fractions and the kinetics of chymosin-induced macropeptide release from carbohydrate-free and carbohydrate-containing fractions determined by high-performance gel-permeation chromatography. Biochem. J. 240:87–97.[Medline]

Wagner, V. A., T. A. Scild, and H. Geldermann. 1994. DNA variants within the 5' flanking region of milk protein-encoding genes II. The ß-Lactoglobulin-encoding gene. Theoret. Appl. Genet. 89:121–126.

Wang Q., J. C. Allen, and H. E. Swaisgood. 1999. Binding of lipophilic nutrients to ß-lactoglobulin prepared by bioselective adsorption. J. Dairy Sci. 82:257–264.[Abstract]

Ward, P. P., S. Uribe-Luna, and O. M. Conneeley. 2002. Lactoferrin and host defense. Biochem. Cell Biol. 80:95–102.[Medline]

Wegrzyn, J. 1978. Allelism of genes determining two IgG₁ allotypes in cattle. Anim. Blood Groups Biochem. Genet 9:59–63.[Medline]

Wei, Z, T. Nishimura, and S. Yoshida. 2000. Presence of a glycan at a potential-glycosylation site, Asn-281, of bovine lactoferrin. J. Dairy Sci. 83:683–689.[Abstract]

Wheelock, J. V., and G. Sinskinson. 1969. Identification of 2-acetamido-2-deoxy-D- galactose in bovine k-casein. Biochim. Biophys. Acta 194:597–599.[Medline]

Wheelock, J. V., and G. Sinskinson. 1973. Carbohydrates of bovine k-casein. J. Dairy Res. 40:413–420.

Whitney, R. M., J. R. Brunner, K. E. Ebner, H. M. Farrell, Jr., R. V. Josephson, C. V. Morr, and H. E. Swaisgood. 1976. Nomenclature of the proteins of cow’s milk: Fourth revision. J. Dairy Sci. 59:795–815.

Wie, S. I., K. J. Dorrington, and A. Froese. 1978. Characterization of the proteolytic fragments of bovine colostral IgG1. J. Immunol. 121:98–104.[Abstract/Free Full Text]

Wijesinha-Bettoni, R., C. M. Dobson, and C. Redfield. 2001. Comparison of the structure and dynamics of holo- and apo--lactalbumin. J. Mol. Biol. 307:885–898.[Medline]

Woychik, J. H. 1964. Polymorphism in -casein of cow’s milk. Biochem. Biophys. Res. Comm. 16:267–271.[Medline]

Woychik, J. H., E. B. Kalan, and M. E. Noelken. 1966. Chromatographic isolation and partial characterization of reduced -casein components. Biochemistry 5:2276–2282.[Medline]

Yan, S.-C. B., and F. Wold. 1984. Neoglycoproteins: In vitro introduction of glycosyl units at glutamines in ß-casein using transglutaminase. Biochemistry 23:3759–3765.[Medline]

Zevaco, C., and B. Ribadeau-Dumas. 1984. A study on the carbohydrate binding sites of bovine -casein using high performance liquid chromatography. Milchwissenschaft 39:206–210.

Zhao, Y., I. Kacskovics, Q. Pan, D. A. Liberles, J. Geli, S. K. Davis, H. Rabbani, and L. Hammarström. 2002. Artiodactyl IgD: The missing link. J. Immunol. 169:4408–4416.[Abstract/Free Full Text]

Zucht, H. D., M. Raida, K. Adermann, H. J. Magert, and W. G. Forssmann. 1995. Casocidin-I: a casein-_S2 derived peptide exhibits antibacterial activity. FEBS Lett. 372:185–188.[Medline]

This article has been cited by other articles:

				C. Weimann, H. Meisel, and G. Erhardt Short communication: Bovine {kappa}-casein variants result in different angiotensin I converting enzyme (ACE) inhibitory peptides J Dairy Sci, May 1, 2009; 92(5): 1885 - 1888. [Abstract] [Full Text] [PDF]

				H. M. Farrell Jr., E. L. Malin, E. M. Brown, and A. Mora-Gutierrez Review of the chemistry of {alpha}S2-casein and the generation of a homologous molecular model to explain its properties J Dairy Sci, April 1, 2009; 92(4): 1338 - 1353. [Abstract] [Full Text] [PDF]

				J. M. L. Heck, A. Schennink, H. J. F. van Valenberg, H. Bovenhuis, M. H. P. W. Visker, J. A. M. van Arendonk, and A. C. M. van Hooijdonk Effects of milk protein variants on the protein composition of bovine milk J Dairy Sci, March 1, 2009; 92(3): 1192 - 1202. [Abstract] [Full Text] [PDF]

				D. J. McMahon and B. S. Oommen Supramolecular Structure of the Casein Micelle J Dairy Sci, May 1, 2008; 91(5): 1709 - 1721. [Abstract] [Full Text] [PDF]

				E.-M. Prinzenberg, H. Jianlin, and G. Erhardt Genetic Variation in the {kappa}-Casein Gene (CSN3) of Chinese Yak (Bos grunniens) and Phylogenetic Analysis of CSN3 Sequences in the Genus Bos J Dairy Sci, March 1, 2008; 91(3): 1198 - 1203. [Abstract] [Full Text] [PDF]

				A. Caroli, S. Chessa, F. Chiatti, D. Rignanese, B. Melendez, R. Rizzi, and G. Ceriotti Short Communication: Carora Cattle Show High Variability in {alpha}s1-Casein J Dairy Sci, January 1, 2008; 91(1): 354 - 359. [Abstract] [Full Text] [PDF]

				B. L. Luhovyy, T. Akhavan, and G. H. Anderson Whey Proteins in the Regulation of Food Intake and Satiety J. Am. Coll. Nutr., December 1, 2007; 26(6): 704S - 712S. [Abstract] [Full Text] [PDF]

				S. Lamothe, G. Robitaille, D. St-Gelais, and M. Britten Short Communication: Extraction of {beta}-Casein from Goat Milk J Dairy Sci, December 1, 2007; 90(12): 5380 - 5382. [Abstract] [Full Text] [PDF]

				A. F. Keating, P. Davoren, T. J. Smith, R. P. Ross, and M. T. Cairns Bovine {kappa}-Casein Gene Promoter Haplotypes with Potential Implications for Milk Protein Expression J Dairy Sci, September 1, 2007; 90(9): 4092 - 4099. [Abstract] [Full Text] [PDF]

				E. M. Ibeagha-Awemu, E.-M. Prinzenberg, O. C. Jann, G. Luhken, A. E. Ibeagha, X. Zhao, and G. Erhardt Molecular Characterization of Bovine CSN1S2*B and Extensive Distribution of Zebu-Specific Milk Protein Alleles in European Cattle J Dairy Sci, July 1, 2007; 90(7): 3522 - 3529. [Abstract] [Full Text] [PDF]

				S. Chessa, F. Chiatti, G. Ceriotti, A. Caroli, C. Consolandi, G. Pagnacco, and B. Castiglioni Development of a Single Nucleotide Polymorphism Genotyping Microarray Platform for the Identification of Bovine Milk Protein Genetic Polymorphisms J Dairy Sci, January 1, 2007; 90(1): 451 - 464. [Abstract] [Full Text] [PDF]

				M. H. Braunschweig and T. Leeb Aberrant low expression level of bovine beta-lactoglobulin is associated with a C to A transversion in the BLG promoter region. J Dairy Sci, November 1, 2006; 89(11): 4414 - 4419. [Abstract] [Full Text] [PDF]

				W. L. Chen, W. T. Liu, M. C. Yang, M. T. Hwang, J. H. Tsao, and S. J. T. Mao A novel conformation-dependent monoclonal antibody specific to the native structure of beta-lactoglobulin and its application. J Dairy Sci, March 1, 2006; 89(3): 912 - 921. [Abstract] [Full Text] [PDF]

				T. L. Mikkelsen, H. Frokiaer, C. Topp, F. Bonomi, S. Iametti, G. Picariello, P. Ferranti, and V. Barkholt Caseinomacropeptide Self-Association is Dependent on Whether the Peptide is Free or Restricted in {kappa}-Casein J Dairy Sci, December 1, 2005; 88(12): 4228 - 4238. [Abstract] [Full Text] [PDF]

				E. L. Malin, E. M. Brown, E. D. Wickham, and H. M. Farrell Jr. Contributions of Terminal Peptides to the Associative Behavior of {alpha}s1-Casein J Dairy Sci, July 1, 2005; 88(7): 2318 - 2328. [Abstract] [Full Text] [PDF]

				D. L. Van Hekken, M. H. Tunick, and Y. W. Park Effect of Frozen Storage on the Proteolytic and Rheological Properties of Soft Caprine Milk Cheese J Dairy Sci, June 1, 2005; 88(6): 1966 - 1972. [Abstract] [Full Text] [PDF]

				A. W. Kuss, J. Gogol, H. Bartenschlager, and H. Geldermann Polymorphic AP-1 Binding Site in Bovine CSN1S1 Shows Quantitative Differences in Protein Binding Associated with Milk Protein Expression J Dairy Sci, June 1, 2005; 88(6): 2246 - 2252. [Abstract] [Full Text] [PDF]

				L. K. Creamer, H. C. Nilsson, M. A. Paulsson, C. J. Coker, J. P. Hill, and R. Jimenez-Flores Effect of Genetic Variation on the Tryptic Hydrolysis of Bovine {beta}-Lactoglobulin A, B, and C J Dairy Sci, December 1, 2004; 87(12): 4023 - 4032. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Farrell, H. M. Articles by Swaisgood, H. E. Search for Related Content PubMed PubMed Citation Articles by Farrell, H. M., Jr. Articles by Swaisgood, H. E.