Characterization of Binding of Transforming Growth Factor-ß1 to Bovine Mammary Membranes

¹ Department of Animal and Food Sciences, University of Vermont, Burlington 05405

This study developed a procedure to measure binding of transforming growth factor-ß1 to bovine mammary membranes. Mammary membranes were incubated with 3 M MgCl₂ to remove endogenously bound transforming growth factor-ß1. Binding was optimized by incubation of 200 µg of membrane protein with ¹²⁵I-labeled transforming growth factor-ß1 in 25 mM Tris, 10 mM CaCl₂, and 1.0% BSA in a total volume of .5 ml in the presence or absence of unlabeled transforming growth factor-ß1. The reaction equilibrated in 2 h at 4°C. Specific binding was linear from 142 to 1140 µg of membrane protein. The reaction was specific for the beta transforming growth factors; transforming growth factor-ß1, transforming growth factor-ß2, and transforming growth factor-ß3 could compete effectively with ¹²⁵I-labeled transforming growth factor-ß1 for the receptor. The growth factors, epidermal growth factor, IGF-I, or transforming growth factor-a did not compete effectively with ¹²⁵I-labeled transforming growth factor-ß1 for binding to bovine mammary membranes. Scatchard analysis showed that the number of receptors averaged 251 pmol/mg of membrane protein and the affinity was 8.7 x l0^-11 M. Binding to mammary membranes was higher during the prepubertal and pubertal periods than during lactation. Binding to mammary membranes during early lactation averaged 24% of the binding observed during other physiological states. Therefore, transforming growth factor-ß1 binds specifically to bovine mammary tissue and varies with stages of development and differentiation, indicating that the transforming growth factor-ß1 receptors may play a role in regulating the activity of transforming growth factor-ß1 in the mammary gland.

Key Words: transforming growth factor-ßbeta; • receptors • bovine • mammary membranes

Submitted on October 14, 1994
Accepted on February 9, 1995

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