Corticoid Binding in Mammary Tissue Slices from Lactating Cows

Abstract

Mammary tissue slices (from lactating Holstein cows) incubated at 37 C with various concentrations of either hydro- gen-3 cortisol or hydrogen-3 dexameth- asone bound both hormones with high affinity (dissociation constants (Kd) -- 10 -10M) . There were approximately 2900 and 3800 total high affinity binding sites per mammary cell for cortisol and dexamethasone, respectively. In addition, a major nonspecific component bound cortisol and dexamethasone and was un- saturable. Unlabeled cortisol and dexa- methasone reduced binding of hydro- gen-3 cortisol and dexamethasone where- as unlabeled 1713-estradiol, testosterone, and progesterone were without effect. Incubation of tissue slices at 4 C reduced the total number of high affinity binding sites for cortisol by about 56% compared with similar measurements at 37 C. How- ever, dissociation constants for the high affinity components were similar at both temperatures (Kd ~ 10d0M). Macro- molecules which specifically bound corti- sol in the 700 × g supernatant and precipitate tissue fractions were isolated by gel filtration chromatography. En- zyme digestion experiments and treat- ment with p-chloromercuribenzoate indi- cated that the macromolecules binding cortisol were proteins. Thin-layer chroma- tography of bound hydrogen-3 cortisol in the 700 × g supernatant indicated that the majority of bound radioactivity was authentic cortisol. We conclude that fresh