Characterization of the purified molybdate‐stabilized glucocorticoid receptor from rat liver

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Abstract
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 11800‐fold purification. The dissociation rate constant of this complex was 1.6 × 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 90000‐Mr band. Three minor bands with Mr of 78000, 72000 and 48000 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 90000‐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°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°C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium.

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