Structure, dissociation, and proteolysis of mammalian steroid receptors. Multiplicity of glucocorticoid receptor forms and proteolytic enzymes in rat liver and kidney cytosols.
The forms of steroid receptors detected in mammalian tissue cytosols vary from a globular fragment, the mero-receptor, with a molecular weight (Mr) of approximately 23,000 to highly asymmetric molybdate-stabilized complexes (Mr approximately 330,000). Our objectives were to investigate the relationships among various receptor forms and mechanisms of stabilization by Na2MoO4, to characterize endogenous proteolytic enzymes, and to evaluate the effects of freezing the tissues on receptor structure and protease activities in the resultant cytosols. Glucocorticoid receptors and proteases were analyzed in cytosols from fresh and frozen rat liver and kidney. Enzymes were assayed fluorometrically at approximately 24 degrees C, with peptidyl tyrosine, lysine, and phenylalanine derivatives of 7-amino-4-methylcoumarin. Rates of cleavage of the tyrosine-containing substrate by crude and partially purified liver cytosol enzymes were markedly suppressed by Na2MoO4. While receptor cleavage by these enzymes has not been demonstrated, these results illustrate the direct inhibition of proteolysis by molybdate. Rates of cleavage of the lysine-containing substrate were 7- to 40-fold higher in cytosols from frozen liver and fresh or frozen kidney, in which the Stokes radius (Rs) of the largest receptor form was 70-74 A, than in fresh liver cytosol, in which Rs was 84 +/- 2 A (n = 20). Filtration of the 84 A complex (Mr approximately 330,000) in hypertonic buffer without Na2MoO4 revealed a 7:3 mixture of forms with Rs of 50-60 A (Mr approximately 90,000) and Rs of 30-40 A (Mr approximately 50,000). The latter are derived from the 50-60 A forms by proteolysis and/or dissociation. We conclude that the 84 A (untransformed) receptor is an oligomer, probably a tetramer, containing the 50-60 A subunits and that purification of the intact structure will require removal or inactivation of contaminating proteases.
D000818:Animals; D002850:Chromatography, Gel; D005470:Fluorometry; D007668:Kidney / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D008982:Molybdenum / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D014222:Triamcinolone Acetonide / Q000378:metabolism
D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D011189:Potassium Chloride; D008982:Molybdenum; D014222:Triamcinolone Acetonide