Corticoid Binding in Mammary Tissue Slices from Lactating Cows

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Abstract
Mammary tissue slices (from lactating Holstein cows) incubated at 37 C with various concentrations of either hydrogen-3 cortisol or hydrogen-3 dexamethasone bound both hormones with high affinity [dissociation constants (Kd) approximately equal to 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 unsaturable. Unlabeled cortisol and dexamethasone reduced binding of hydrogen-3 cortisol and dexamethasone whereas unlabeled 17beta-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. However, dissociation constants for the high affinity components were similar at both temperatures (Kd approximately equal to 10-10M). Macromolecules which specifically bound cortisol in the 700 X g supernatant and precipitate tissue fractions were isolated by gel filtration chromatography. Enzyme digestion experiments and treatment with p-chloromercuribenzoate indicated that the macromolecules binding cortisol were proteins. Thin-layer chromatography of bound hydrogen-3 cortisol in the 700 X g supernatant indicated that the majority of bound radioactivity was authentic cortisol. We conclude that fresh mammary tissue from lactating cows possesses protein which specifically binds corticoids with high affinity.