CYTOSOL AND NUCLEAR [3H]OESTRADIOL BINDING IN THE FOETAL TISSUES OF GUINEA PIG

Abstract

The formation of [3H]estradiol macromolecule complexes was studied in vivo and in vitro in kidney, lung and liver of intact fetal guinea pig. Specific binding of [3H]estradiol was demonstrated in the cytosol and nuclear fractions of fetal kidney. Intensive binding was found in the cytosol of fetal lung, but most of the bound radioactive material (71%) was [3H]estrone. Some binding was found in the cytosol but not in the nucleus of fetal liver. In in vivo experiments, the binding of radioactive material to plasma proteins was studied: 22% of the plasma radioactivity was bound, of which 67% was identified as estrone sulfate. Estradiol sulfate represented 7% and unconjugated estradiol 0.6% (0.1% of the total plasma radioactivity). Of fetal plasma radioactivity, 2-3% was unbound [3H]estradiol. The formation of [3H]estradiol complexes in the kidney cytosol fraction does not depend on temperature, while nuclear [3H]estradiol complexes increase with increasing temperature. Maximal formation of [3H]estradiol complexes in the cytosol fraction and the 0.1 M Tris and 0.3 M NaCl nuclear extracts was reached after 15 min, but binding in the 1 M NaCl nuclear extract continued to increase up to 30 min. After incubation of purified nuclei of fetal kidney with 1.1 .times. 10-7 M [3H]estradiol, specific binding was found in the different nuclear fractions. Specific binding was detected in isolated nuclei previously extracted by 0.1 M Tris and 0.3 M NaCl before being incubated with 1.1 .times. 10-7 M [3H]estradiol. The Kd of binding of [3H]estradiol in the renal cytosol fraction is 2.5 .times. 10-10 M with n = 4.5 .times. 10-14 mol/mg protein. Incubation of isolated 1 M NaCl nuclear extract from fetal kidney with [3H]estradiol gives a Kd of 3.3 .times. 10-10 M with n = 2.5 .times. 10-14 mol/mg protein. The nuclear complexes in the fetal kidney could be formed either through an intermediate cytosol complex, or the 2 step mechanism could take place in the nucleus. Direct binding of [3H]estradiol with high affinity was observed in the 1 M NaCl nuclear extract.