Progestin binding in mammary tissue of prepartum, nonlactating and postpartum, lactating cows

Binding of [3H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-=dione) to bovine mammary cytosol indicated the presence of progestin binding sites of high-affinity and low-capacity in tissue from prepartum, nonlactating and from postpartum, lactating cows. To prevent binding of [3H]R5020 to glucocorticoid binding sites, a 200-fold molar excess of nonradioactive cortisol was included during all incubations, thus specific binding was limited to progestin binding sites. Nonradioactive R5020 and progesterone effectively inhibited [3H]R5020 binding to progestin binding sites, while estradiol-17 beta, dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), dexamethasone (9-fluoro-11 beta, 17, 21-trihydroxy-16 alpha methyl-1,4-pregnadiene-3,20-dione) or additional cortisol were ineffective. Dissociation constants for especially bound [3H]R5020 in cytosol from mammary tissue of nonlactating and lactating cows were nearly identical, averaging 1.9 (+/- 0.3) and 0.8 (+/- 0.2) x 10(-9)M, respectively. However, binding capacities (fmol/mg cytosolic protein) were greater in cytosol from prepartum, nonlactating (179 +/- 53) than postpartum, lactating (41 +/- 15) cows. Specific binding components in cytosol from lactating cows sedimented iun the 6-7S region on linear sucrose density gradients. When subjected to isoelectric focusing, specific binders with isoelectric points (pI) of approximately 6.1, 7.9 and 8.3 were resolved. The decrease in number of binding sites during lactation was due to the virtual absence of the anionic binding species, suggesting that their presence is necessary for progesterone to inhibit milk secretion.