Characterization of Binding of Transforming Growth Factor-β1 to Bovine Mammary Membranes

Abstract

This study developed a procedure to measure binding of transforming growth factor-β1 to bovine mammary membranes. Mammary membranes were incubated with 3 M MgCl2 to remove endogenously bound transforming growth factor-β1. Binding was optimized by incubation of 200 μg of membrane protein with 125I-labeled transforming growth factor-β1 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-β1. 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-β1, transforming growth factor-β2, and transforming growth factor-β3 could compete effectively with 125I-labeled transforming growth factor-β1 for the receptor. The growth factors, epidermal growth factor, IGF-I, or transforming growth factor-α did not compete effectively with 125I-labeled transforming growth factor-β1 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 × 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-β1 binds specifically to bovine mammary tissue and varies with stages of development and differentiation, indicating that the transforming growth factor-β1 receptors may play a role in regulating the activity of transforming growth factor-β1 in the mammary gland. © 1995, American Dairy Science Association. All rights reserved.