Analysis of binding of [3H]Estradiol to the cytosol fraction of rat pancreas: comparison with sites in the cytosol of uterus.

The cytosol fraction of rat pancreas [100,000 X g (for 1 h) supernatant] demonstrated specific but nonsaturable binding of [3H]estradiol in the concentration range of 2-50 X 10(-9) M. Scatchard analysis of specifically bound [3H]estradiol, determined by the isotope dilution technique (competition with excess unlabeled estradiol), indicated a single class of binding sites (approximately 4.4 pmol/mg protein) with an apparent Kd of 5 X 10(-8) M. Such cytosol fractions, prepared from homogenates that contained protease inhibitors, when prelabeled with [3H]estradiol, demonstrated a single sharp eluate peak of radioactivity after Sephadex G-200 chromatography that corresponded to a molecular weight of 120,000. When protease inhibitors were omitted, [3H]estradiol was associated with material of considerably lower molecular weight. Routinely, the protease inhibitors leupeptin (1 mM), phenylsulfonyl fluoride (0.5 mM), and tosylphenylalanylchloromethyl ketone (0.05 mM) were included in the buffer used for homogenizing both the pancreas and uterus. The protein that binds [3H]estradiol in uterus differed from that in pancreas in a number of ways: 1) in the range of 10-20 X 10(-9) M [3H]estradiol, specific binding of the hormone to uterine sites was saturable, and Scatchard analysis indicated a single class of binding sites, (approximately 0.5 pmol/mg protein) having an apparent Kd of 3 X 10(-10) M; 2) the rate constants of dissociation of the [3H] estradiol-bound complexes in pancreas and uterus were 3.1 X 10(-4) sec-1 (t1/2, 37 min) and 8.7 X 10(-5) sec-1 (t1/2, 134 min), respectively; 3) the molecular weight of the estrogen-binding protein in freshly prepared uterine supernatant fractions appeared to be at least 240,000; this was unaltered regardless of whether protease inhibitors were present during initial homogenization of the tissue; and 4) when uterine supernatants prepared in the absence of protease inhibitors were kept at 8 C for 24 h and then analyzed by Sephadex G-200 chromatography, a second peak of [3H]estradiol-binding activity appeared at the same eluate volume as the low molecular weight binding fractions of pancreas. These data suggest that although the binding proteins in pancreas and uterus are different, there may be some common features at the hormone-binding locus.