ISOLATION AND PROPERTIES OF CORTISOL METABOLITE BINDING PROTEINS OF RAT LIVER CYTOSOL

Two proteins which bind the two most negatively charged metabolites of radioactive cortisol formed in 45 min were isolated from rat liver cytosol. Protein-bound radioactivity was separated initially from unbound radioactivity by column chromatography on Bio-Gel P-100. It then was fractionated into two protein-radioactivity complexes upon chromatography on DEAE-Sephadex A-50. Each binding protein was purified to homogeneity. The large binder was purified by chromatography on Cellex-phosphate followed by Sephadex G-75. The small binder was purified by chromatography and rechromatography on Sephadex G-75. The large binder had an estimated molecular weight of 37,000 calculated from amino acid analysis and from sedimentation velocity and diffusion data, although a value of 50,000 ± 6,000 was obtained by chromatography on Sephadex G-75. The sedimentation coefficient was 3.47 S, a value apparently independent of protein concentration and the isoelectric point was at pH 8.9. It was homogeneous in the analytical ultracentrifuge, by moving-boundary electrophoresis, by column chromatography, and by pH electrofocusing. The small binder had a molecular weight of 6950 calculated from amino acid analysis and 4000-5000 by chromatography on Sephadex G-75. It had a low tyrosine content and an absorbance maximum at 260 mμ suggesting the presence of nucleotides. The isoelectric point was at pH 5.4. It was homogeneous by column chromatography and by pH electrofocusing. The cortisol metabolite binders were specific in binding. The large binder had only the most negatively charged metabolite of cortisol associated with it, while the small binder was bound to the metabolite of intermediate negative charge. The third metabolite of cortisol, with the least anionic charge did not appear to be bound to proteins. © 1969, American Chemical Society. All rights reserved.