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Abstract
Abstract Of four protein fractions previously reported which bind corticosteroids in liver cytosol, one fraction, corticosteroid Binder II, has been found to be a major hormone receptor. It has a molecular weight of 67,000 determined by gel filtration. Binder II has an appropriate glucocorticoid binding specificity: dexamethasone g corticosterone g cortisol >> cortisone >> deoxycorticosterone and it also binds progesterone and to a lesser extent the sex hormones. The Kd for dexamethasone is about 6 x 10-10 m determined in vitro and that for cortisol is in the range of 10-8 m. Binder II bound with corticosteroid is deduced to undergo nuclear transfer in vitro and in vivo as determined by its reisolation from the soluble nuclear fraction. The reisolated nucleoplasmic macromolecule has characteristics similar to corticosteroid Binder II isolated from liver cytosol (molecular weight, chromatographic elution position and pI (6.7) determined by isoelectric focusing). An antibody has been prepared to corticosteroid Binder II fraction isolated from cytosol which does not cross-react with other corticosteroid binding proteins (ligandin, Binder III and transcortin) and an antigen, determined by gel immunodiffusion, is present in the soluble nuclear fraction after nuclear transfer of macromolecular bound steroid in vitro or in vivo.
Highlights
A molecular weight of 67,000 for the binding protein was determined on Sephadex
We calculated that Binder II was concentrated to 0.005% of the liver cytosol proteins [4] based on recovery of protein and correcting for losses of total bound radioactivity
Corticost,eroid Binder II clearly has been shown to constitute a major part of the liver hormone receptor function. It possesses appropriate binding specificity, association constant, molecular and biological properties including nuclear transfer. It has been reisolated from nuclear soluble fraction following nuclear transfer and shown to have the same properties as Binder II isolated from the cytosol
Summary
Anima&-Adrenalectomized male CD Fisher rats (110 to 140 g; Charles River 13reeding Labs) were used throughout these experiments 7 to 10 days following surgery. They were fed a normal chow diet and 0.1% NaCl to drink. Postmortem examination showed no residual adrenal tissue. For immunological studies on certain tissue cytosols, intact female Sprague-. Dawley rats (Holzmann) weighing 250 g were used. When livers were taken for experiments, they were first perfused in situ with sterile 0.9% NaCl solution through the portal vein. Livers were removed and homogenized in 0.25 M sucrose-50 mM Tris-HCl, pH 7.5 [10]
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