Structure of the mouse glucocorticoid receptor: rapid analysis by size-exclusion high-performance liquid chromatography

Abstract

Gel-exclusion high-performance liquid chromatography (HPLC) has been used to separate the untransformed from the transformed glucocorticoid receptor (GC-R) extracted from mouse AtT-20 cells. With 200 mM potassium phosphate as the eluent, an efficient separation of the forms of the GC-R is attained in 15-20 min. The untransformed cytosolic GC-R elutes from the column with a Stokes radius (Rs) of 8.2-8.6 nm, as do the molybdate-stabilized GC-R, the purified untransformed GC-R, and the cross-linked cytosolic GC-R. GC-R transformed in vitro by either ammonium sulfate precipitation, KCl treatment, or G-25 chromatography elutes with an Rs of 5.7-6 nm. Also, GC-R extracted from the nucleus with either 0.3 M KCl or 2 mM sodium tungstate, or purified by two cycles of DNA-cellulose chromatography, has an Rs of 5.5-6.3 nm. The data are identical either in the presence or in the absence of 20 mM Na2MoO4, suggesting that molybdate is not causing aggregation to produce a larger Rs value than that of the native receptor. Vertical tube rotor sucrose gradient ultracentrifugation of cytosol produces three forms of the GC-R: 9.1 S, 5.2 S, and 3.8 S. Sequential analysis of the GC-R forms by HPLC and vertical tube rotor ultracentrifugation and vice versa allows for the hydrodynamic determination of molecular weight within a very short time period (2-3 h total).(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of detectable ribonucleic acid in the purified, untransformed mouse glucocorticoid receptor

“…Protein fractionation and characterization were carried out by HPLC on an Altex TSK-3000SW exclusion column (7.5 X 300 mm) preceded by a TSK-3000SW guard column (7.5 X 100 mm) as described elsewhere (LaPointe et al, 1986). To determine the Stokes radius (7?s) of the GC-R, the column was calibrated with 14C-methylated RNase A (1.64 nm), ovalbumin (2.86 nm), bovine serum albumin (3.59 nm), ferritin (6.15 nm), and thyroglobulin (8.61 nm).…”