Studies on Renal Aldosterone-binding Proteins

Abstract

Abstract 3H-Aldosterone-macromolecular complexes were isolated from the nuclear and cytosol fractions of rat kidneys by precipitation with (NH4)2SO4 and by chromatography on Sephadex G-50 and G-75. These complexes were recovered after injection of 3H-aldosterone into adrenalectomized rats and after incubation of labeled steroid with renal nuclear extracts and cytosol fractions in vitro. The nuclear aldosterone binding substances were identified as proteins by the following criteria: (a) chemical analysis of Tris-CaCl2 extracts of purified nuclear fractions; (b) dissociation of the complexes by wide spectrum proteolytic enzymes but not by nucleases; and (c) loss of 3H-aldosterone-binding activity when the complexes were treated with the sulfhydryl reagents, p-hydroxymercuribenzoate and 2-(2-aminoethyl)-2-thiopseudourea dihydrochloride. The cytosol aldosterone-binding substances were identified as proteins by the following criteria: (a) dissociation of the complexes by wide spectrum proteolytic enzymes but not by nucleases, lipase, or phospholipase; (b) loss of 3H-aldosterone-binding activity on treatment with p-hydroxymercuribenzoate. Estradiol-17β,6α-methylprednisolone, deoxycorticosterone, and 9α-fluorocortisol impaired the formation of the 3H-aldosterone-macromolecular complexes in the nuclear and cytosol fractions in direct proportion to their potencies in the regulation of renal tubular transport of sodium. In the in vitro system, spirolactone inhibited the formation of the 3H-aldosterone complex at concentration ratios that inhibit the action of aldosterone in the rat in vivo. The isomer of aldosterone, 17-isoaldosterone, was not bound to the proteins in nuclear extracts, and at a 10-fold concentration ratio did not inhibit the formation of 3H-aldosterone complexes in nuclear extracts. In addition, 17-isoaldosterone (1.4 x 10-7 m) had no effect on active sodium transport in the isolated toad bladder system and at 1 µg/150 g of body weight had no effect on the urinary Na+:K+ concentration ratios in the adrenalectomized rat. Thus, we have isolated and partially purified renal nuclear proteins which have many of the properties to be expected of the specific mineralocorticoid receptors.