Glucocorticoid Receptor Cleavage by Leupeptin-Sensitive Enzymes in Rat Kidney Cytosol

Summary

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 (R_S=23±3 Å) and the isoelectric point (pI=5.9 at 4°). 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 (pI=4.9 at 4°) and centrifugation in glycerol gradients (s_{20 ,w} = 9–11 S in 50 mM KC1). When this large complex in fresh cytosol was analyzed on Agarose (Bio-Rad) at a high flow rate, it had R_S≃60 Å in 50 mM KC1 and R_S≃30 Å in 400 mM KC1. 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.

Dedicated to the memory of Gerson T. Margolish