Generation of corticosteroid binder IB from binder II by a sulfhydryl dependent renal cytosolic factor.
It has long been debated whether binder IB represents a unique form of the glucocorticoid receptor or is derived from the larger molecular weight form, binder II, by limited proteolysis. Transformed glucocorticoid receptors in kidney, liver and mixed kidney/liver cytosols were examined using anion exchange and gel filtration chromatography. The transformed receptor in liver cytosols chromatographs as binder II on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of approx 6.0 nm. The transformed receptor in kidney cytosols chromatographs as binder IB on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of 3.0-4.0 nm (3.2 nm on agarose; 3.0-4.0 nm on Sephadex G-100). Using cytosols prepared from mixed homogenates (2 g kidney plus 8 g liver tissue), our experiments show that binder II is converted to a lower molecular weight form (Rs = 3.2 nm on agarose; Rx = 3.9 nm on Sephadex G-100) that is identical to binder IB in its elution position from DEAE-Sephadex anion exchange resin. Identical results are obtained using kidney/liver/cytosols mixed in vitro in which only the hepatic receptor, binder II, is labelled with [3H]TA. These results support the hypothesis that the renal receptor, binder IB, is a proteolytic fragment of binder II and does not represent a polymorphic form of the glucocorticoid receptor. The renal converting activity is dependent on free-SH for full activity but is insensitive to the protease inhibitors leupeptin, antipain, and PMSF. The conversion of hepatic binder II to binder IB in in vitro mixing experiments can be prevented if kidney cytosol is gel filtered on Sephadex G-25 and the eluted macromolecular fraction is adjusted to 10 mM EGTA (or EDTA) prior to mixing with the [3H]TA labelled hepatic cytosol.
Liu
K
K
Department of Biology, San Diego State University, CA 92182-0057.
Ellis
J
J
Barnett
C A
CA
D000315:Adrenalectomy; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology*; D007668:Kidney / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*
D011965:Receptors, Glucocorticoid; D010447:Peptide Hydrolases; D005033:Ethylmaleimide