[3H]Dexamethasone Binding by Rat Liver Microsomes: Effects of Age, Sex, and Adrenal Status*

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Abstract
Microsomal preparations isolated from normal rat liver exhibit a high level of specific [3H]dexamethasonebinding activity. Binding proceeds rapidly and under standardized assay conditions is essentially complete within 30 min at 22–23 C, with nonspecific binding (i.e. binding not displaceable by excess nonradioactive steroid) constituting only a small fraction of the total. Scatchard analysis of radioactive dexamethasone binding indicates a single class of binding sites with a Kd of 2.0 ± 0.1 × 1O-7 M and a binding capacity in the range of 5 pmol/ mg protein for microsomes from male rats in the 200- to 250-g weight range. Binding capacity is greater in male than in female rats and rises strikingly with advancing age, whereas binding affinity remains invariant. Binding, moreover, is shown to be strongly dependent upon the presence of intact adrenal secretion, in that adrenalectomy results in at least an 80% fall in specific dexamethasone binding, while replacement with exogenous glucocorticoid restores the binding activity. Binding activity is destroyed by brief exposure of microsomes to Pronase or sulfhydryl-reactive agents such as p-chloromercuiriphenyl sulfonic acid or 5,5′-dithiobis-(2-nitrobenzoic acid). In the latter instance, the loss of binding is reversible, and binding can be restored by subsequent treatment of inactivated preparations with mercaptoethanol. Although the physiological significance of the high level of glucocorticoid-dependent microsomal steroid hormone binding reported in the present studies remains to be determined, possibilites include both binding to enzymatic steroid- metabolizing sites as well as attachment to membrane sites that may play a role in steroid translocation. It is suggested that such binding may, at least in part, account for the impressively high levels of whole cell glucocorticoid binding described in several recent reports, in which binding by cytosol accounts for less than half of the total cellular binding sites and in which the Kd for the particulate fractions, as compared to that for soluble receptors in the cytosol, is similarly greater.