The marginal utility of affinity chromatography using immobilized estradiol in the purification of human uterine estrogen receptor

Abstract

The purification of human uterine estrogen receptor (ER) was performed by way of the affinity chromatography technique using immobilized 17 beta-estradiol on Sepharose 4B, which was developed by Sica and Bresciani (Biochemistry, 18: 2369-2378, 1979). The anti-human ER antibody was induced and its immunological reactivity was also studied. About 300 micrograms of ER were obtained with the recovery of 18%. The purification magnitude of ER was 4000 times. The purified ER appeared as a complex with 17 beta-estradiol, and formed aggregated macromolecule which was eluate at void volume in Sephadex G-200 and Sephacryl S-300 gel filtration. In 7% approximately 18% linear gradient polyacrylamide gel electrophoresis after SDS treatment, two equally stained bands were observed at 90,000 and 70,000 MW areas. Although anti ER antiserum formed no detectable immunoreactive perception with any kinds of antigens, the gamma-globulin fraction of the antiserum produced a precipitate with human and cow uterine cytosols or their purified ERs. On the other hand, two other precipitin lines were observed in human uterine cytosol. One of the lines was also prevalently found with human muscle and liver cytosols, and human whole serum. The immunoreactive component in the human serum was detected in the globulin fraction, but not in the albumin fraction. These facts imply the immunological heterogeneity of the antibody induced by the purified human uterine ER using the affinity chromatography technique. The cytosol of human uterus obtained from hormonally treated women contains lesser amounts of ER than that of cow uterus in Dextran coated charcoal assay. The ER could not fully occupy the estradiol immobilized on Sepharose 4B in the purification of human uterine ER. Therefore, the non-specific estrogen binding proteins originated both from serum and cytoplasm such as steroid binding globulin, could bind to the remaining free estradiol-ligands of the affinity columns. Thus the marginal utility of the affinity chromatography using immobilized estradiol in the purification of human uterine ER was suggested.