Purification of the endogenous glucocorticoid receptor stabilizing factor.
A ubiquitous, low molecular weight, heat-stable component of cytosol stabilizes the glucocorticoid receptor in its untransformed state in association with hsp90. This heat-stable factor mimics molybdate in its effects on receptor function, and it has the heat stability, charge, and chelation properties of a metal oxyanion [Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H., & Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817]. In this paper, we describe the further purification of the endogenous factor from rat liver cytosol by anion-exchange HPLC (Ion-110) after prepurification by molecular sieving, cation absorption, and charcoal absorption. Elution of the factor with an isocratic gradient of ammonium bicarbonate results in recovery of all of the bioactivity in a single peak which coelutes with inorganic phosphate and contains all of the endogenous molybdenum. The bioactivity can be separated from inorganic phosphate by chromatography of the partially purified endogenous factor on a metal-chelating column of Chelex-100. The chelating procedure results in complete loss of bioactivity with recovery of 98% of the inorganic phosphate in both the column drop-through and a subsequent 1 M NaCl wash. The factor preparation purified through the Ion-110 HPLC step inhibits temperature-mediated dissociation of the immunopurified glucocorticoid receptor-hsp90 complex, but it is considerably more effective at stabilizing the unpurified receptor-hsp90 complex in a Chelex-treated cytosol system that has been depleted of metal components. These observations support the proposal that an endogenous metal can stabilize the binding of hsp90 to the receptor but it is likely that other cytosolic components that are not present in the immunopurified complex must contribute to the stability of the soluble protein-protein complex in cytosol.
Meshinchi
S
S
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
Stancato
L F
LF
Gordon
B M
BM
Jones
K W
KW
Pratt
W B
WB
D000818:Animals; D001685:Biological Factors / Q000737:chemistry / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000737:chemistry; D006358:Hot Temperature; D008099:Liver / Q000737:chemistry; D008982:Molybdenum; D010710:Phosphates / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000378:metabolism*
D001685:Biological Factors; D010710:Phosphates; D010743:Phospholipids; D011965:Receptors, Glucocorticoid; D008982:Molybdenum