High-performance chromatofocusing of steroid receptor proteins in the presence and absence of steroid. Investigation of steroid-dependent alterations in surface charge heterogeneity.
We have previously reported the development of high-performance chromatofocusing (HPCF) systems for rapid evaluation of the surface charge heterogeneity of steroid receptor proteins, each in the presence of its specific steroid ligand. However, the surface charge properties of ligand-free receptor proteins remain largely unknown. We have now employed HPCF to rapidly evaluate the surface charge properties of cytosolic estrogen receptor proteins in both the presence and absence of the ligand ([3H]estradiol-17 beta). All operations were performed at 0-4 degrees C. Cytosols prepared from immature calf uteri were preparatively analyzed by HPCF on a SynChropak AX-500 column (25 cm X 4.6 mm I.D.) either before or after incubation with 5-10 nM [3H]estradiol-17 beta. Elution of receptor was by generation of internal pH gradients (pH 8.1 to 3.2) using Pharmacia Polybuffers 96 and 74. Postcolumn detection of previously unliganded receptor was accomplished by incubation of pH-neutralized (pH 7.4) fractions with 5 nM [3H]estradiol-17 beta in the presence and absence of unlabelled competitor. Specifically bound steroid was determined in each fraction using an hydroxylapatite adsorption assay. Significant surface charge heterogeneity was observed for both unliganded receptor and the steroid-receptor complex. The heterodisperse pattern of receptor surface charge appeared to vary in a steroid-dependent manner. Preformed steroid-receptor complexes eluted primarily between pH 6.5-7 and between pH 5-6, with indications for heterogeneity within both regions. The surface charge distribution of unliganded receptor routinely revealed additional, more acidic eluting (pH 3.8-4.6) receptor forms. Sodium molybdate, a commonly used receptor-stabilizing agent, maintains receptors during HPCF as relatively acidic eluting forms (pH 3.8-5.0). The specific elution profile of molybdate-stabilized receptor also appears steroid-dependent. These data demonstrate that HPCF can be used preparatively to rapidly isolate unliganded receptor forms in a biologically active state.
Hutchens
T W
TW
Dunaway
H E
HE
Besch
P K
PK
D000327:Adsorption; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000032:analysis; D017886:Durapatite; D005260:Female; D006882:Hydroxyapatites; D007525:Isoelectric Focusing; D011506:Proteins / Q000032:analysis; D011960:Receptors, Estrogen; D011987:Receptors, Steroid / Q000032:analysis*; D014599:Uterus / Q000032:analysis
D002352:Carrier Proteins; D006882:Hydroxyapatites; D011506:Proteins; D011960:Receptors, Estrogen; D011987:Receptors, Steroid; C010140:estrophilin; D017886:Durapatite