Structure, dissociation, and proteolysis of mammalian steroid receptors. Multiplicity of glucocorticoid receptor forms and proteolytic enzymes in rat liver and kidney cytosols.
1983; Elsevier BV; Volume: 258; Issue: 17 Linguagem: Inglês
10.1016/s0021-9258(17)44466-8
ISSN1083-351X
AutoresMerry R. Sherman, Mary C. Moran, Fe B. Tuazon, Yee-Wan Stevens,
Tópico(s)Hormonal and reproductive studies
ResumoThe forms of steroid receptors detected in mammalian tissue cytosols vary from a globular fragment, the mero-receptor, with a molecular weight (Mr) of "23,000 to highly asymmetric molybdate-stabilized complexes (Mr -330,000).Our objectives were to investigate the relationships among various receptor forms and mechanisms of stabilization by NazMo04, 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 -24 "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 NazMoO,.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-?4 A , than in fresh liver cytosol, in which Rswas 84 f 2 A (n = 20).Filtration of the 84 A complex (M.-330,000) in hypertonic buffer without NazMo03 revealed a 7:3 mixture of formse with Rs of 50-60 A (M. -90,000) and Rsof 30-40 A (vr -50,000).The latter are derived from the 50-60 A forms by prote; olysis 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 re- moval or inactivation of contaminating proteases.The diversity of concepts about the sizes and structures of mammalian steroid receptors is quite remarkable.Molecular
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