Glucocorticoid receptor cleavage by leupeptin-sensitive enzymes in rat kidney cytosol.

The proteolytic origin of small fragments of both the glucocorticoid and mineralocorticoid receptors in rat kidney cytosol was inferred from the effects of leupeptin, a bacterial tripeptide that inhibits many proteases [Sherman, M.R. et al., (1978). Federation Proc. 37:167--173]. In the present study, the smallest fragment of the glucocorticoid receptor containing the steroid-binding site, the mero-receptor, was characterized with respect to the Stokes radius (RS = 23 +/- 3 A) and the isoelectric point (pI = 5.9 at 4 degrees). Chromatography of cytosol labeled with [3H]triamcinolone acetonide on Sephadex LH-20 (Pharmacia) in aqueous buffer resolved the steroid-receptor complex from the unmodified free steroid and from steroid metabolites and contaminants. This technique facilitated analyses of the leupeptin-stabilized receptor form by isoelectric focusing (pU = 5.9 at 4 degrees) and centrifugation in glycerol gradients (s20,w = 9--11 S in 50mM KCl). When this large complex in fresh cytosol was analyzed on Agarose (Bio-Rad) at a high flow rate, it had RS congruent to 60 A in 50 mM KCl and RS congruent to 30 A in 400 mM KCl. These analytical studies with leupeptin indicate the need for inexpensive, irreversible inhibitors of proteolytic enzymes for the purification of intact receptors, holo-receptors, from kidney and other tissues. Specific proteases can then be applied to dissect the holo-receptor into the globular mero-receptor, proximal to the steroid-binding site, and the asymmetric region(s), distal segment(s), that may be involved in the nuclear interactions.