Kinetic characterization of [125I] iodo-prolactin in binding to primary monolayer cultures of rabbit mammary epithelium.
Monocellular suspensions of epithelial cells from mammary glands of rabbits at 20-22 days of pregnancy were prepared by sequential dissociation with collagenase-hyaluronidase followed by Pronase. Maintenance in D-valine-substituted minimum essential medium (D-valine-MEM) supplemented with 10% dialyzed calf serum yielded monolayers enriched for rabbit mammary epithelial cells (RMEC). RMEC specifically and reversibly bound bovine PRL with Ka = 1.41-1.85 x 10(9)M-1. Association of lactogen with RMEC receptor followed bimolecular reaction kinetics with rate of 5.17 (+/- 0.75) x 10(5)M-1 sec-1 at 24 C, and 1.03 (+/- 0.11) x 10(6)M-1 sec-1 at 37 C. Dissociation was first order (K-1 = 5.97 (+/- 0.70) x 10(-5) sec-1) and was unaffected by the presence of lactogen. Specific binding determined with an excess of unlabelled bPRL was 66-77% of the total binding, and was optimal at pH 7.4. The binding reaction reached equilibrium in 2 h at 37 C, in 3 h at 24 C, and after 24 h at 4 C. Studies of binding capacity revealed the presence of 4.6-6.3 x 10(3) sites per cell, competition for which was limited to hormones demonstrating lactogenic activity. Recovered lactogen was not degraded by incubation with or dissociation from RMEC. Approximately 25% of the radioactivity remained associated with the cells even upon prolonged incubation. These studies demonstrated several advantages of RMEC for the investigation of hormone-receptor interaction and receptor regulation.
D000818:Animals; D002469:Cell Separation; D002478:Cells, Cultured; D003470:Culture Media; D004848:Epithelium / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000166:cytology / Q000378:metabolism*; D011247:Pregnancy; D011388:Prolactin / Q000378:metabolism*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011981:Receptors, Prolactin
D003470:Culture Media; D011956:Receptors, Cell Surface; D011981:Receptors, Prolactin; D011388:Prolactin