Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex.
Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three-step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high-performance size-exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone-acetonide-receptor complex was obtained in 20% yield with an overall 11 800-fold purification. The dissociation rate constant of this complex was 1.6 X 10(-4) min-1. The purified receptor sedimented at 8.3 S in high-salt and 9.4 S in low-salt sucrose gradients containing molybdate. A 7.0-nm Stokes radius was determined by HPSEC on a TSK G 4000 column in high-salt buffer. The calculated Mr was 278000. Dodecyl sulfate/polyacrylamide gel electrophoresis revealed an almost homogeneous 90 000-Mr band. Three minor bands with Mr of 78 000, 72 000 and 48 000 were also inconstantly seen. An apparent pI = 5.1 was observed for the [3H]steroid complex by isoelectric focusing in agarose gel. Furthermore high-performance ion-exchange chromatography of the purified complex on a DEAE 545 LKB column (DEAE HPLC) yielded a sharp peak eluted at a 315 mM potassium ion concentration. This peak was shown to contain almost all the 90 000-Mr protein. Moreover the purified receptor complex appeared to be transformable to a DNA-binding state after molybdate removal followed by warming 30 min at 25 degrees C in presence of 0.2% bovine serum albumin: 50-78% transformation yield could be demonstrated by DNA-cellulose chromatography. Partial transformation could also be obtained at 0 degrees C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium.
D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000379:methods; D004247:DNA / Q000031:analogs & derivatives; D003907:Dexamethasone; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D008099:Liver / Q000378:metabolism*; D008982:Molybdenum; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification*; D014222:Triamcinolone Acetonide
C033190:DNA-cellulose; D011965:Receptors, Glucocorticoid; C044659:molybdate; D003907:Dexamethasone; D008982:Molybdenum; D002482:Cellulose; D004247:DNA; D014222:Triamcinolone Acetonide