Comparison of glucocorticoid-binding proteins in normal and neoplastic mammary tissues of the rat.
Kinetic and molecular properties of components binding [3H]triamcinolone acetonide were studied using 105,000g supernatants of lactating mammary gland, R3230AC, and dimethylbenz[a]anthracene (DMBA) induced mammary tumors of the rat. Using a dextran-coated charcoal adsorption procedure, the relationship between specific glucocorticoid binding and protein concentration was linear in the range of 0.5-4.0 mg/reaction. These cytoplasmic macromolecules bound [3H]triamcinolone acetonide with limited capacity (50-400 fmol/mg of cytosol protein) and high affinity, Kd approximately 10(-8)-10(-9) M. Optimal binding was obtained when homogenizations were made in Tris buffers, at pH 7.4, containing monothioglycerol. Time course of association of [3H]triamcinolone acetonide and its binding sites showed maximal binding by 6-8 hr at 3 degrees which remained unchanged up to 24 hr. The rate constant of association at 3 degrees was in the range of 2-4 x 10(5) M-1 min-1. The rate constant of dissociation of bound [3H]triamcinolone acetonide could not be calculated accurately since the reaction was essentially irreversible for 5 hr at 3 degrees. Estimation of the half-life of the steroid-binding protein complexes from the Kd and the rate constant for association gave a value of 11-12 hr. From ligand specificity studies, the glucocorticoids, triamcinolone acetonide, corticosterone, cortisol, and dexamethasone competed well for [3H]triamcinolone acetonide binding sites. Progesterone, aldosterone, and the anti-glucocorticoid, cortexolone, were also good competitors while androgens and estrogens were weak inhibitors of binding. The binding compenents sedimented at 7-8 S in sucrose gradients of low ionic strength and dissociated into lower molecular weight components sedimenting at 4-5S in high ionic strength gradients. Studies in vivo using animals bearing the DMBA-induced tumor demonstrated that [3H]triamcinolone acetonide binding complexes were present in cytoplasmic and nuclear compartments. Sedimentation coefficients of the cytoplasmic and nuclear forms of these receptors labeled in vivo were 7-8S and 4-5S, respectively. These studies suggest that the molecular and kinetic binding properties of glucocorticoid receptors in neoplastic mammary tissues are similar to those of the normal mammary gland.
D015127:9,10-Dimethyl-1,2-benzanthracene; D000818:Animals; D001667:Binding, Competitive; D002021:Buffers / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007774:Lactation; D008321:Mammary Glands, Animal / Q000378:metabolism*; D008325:Mammary Neoplasms, Experimental / Q000139:chemically induced / Q000378:metabolism*; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000378:metabolism; D011247:Pregnancy; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013997:Time Factors; D014222:Triamcinolone Acetonide / Q000378:metabolism*
D002021:Buffers; D009363:Neoplasm Proteins; D011506:Proteins; D011956:Receptors, Cell Surface; D013439:Sulfhydryl Reagents; D015127:9,10-Dimethyl-1,2-benzanthracene; D014222:Triamcinolone Acetonide