Characterization of a Proposed Oxytocin Receptor in Rat Mammary Gland

Abstract

Abstract [3H]Oxytocin, incubated with mammary tissue from the lactating rat, was bound specifically to three particulate fractions obtained by homogenization and centrifugation at 1,000, 20,000, and 105,000 x g. Radioactivity in the cytosol appeared to be unbound oxytocin, as determined by gel filtration on Sephadex G-100. Oxytocin was distributed about equally among the three particulate fractions and could not be correlated with either the amount of protein or the enzymes putatively characteristic of cell membranes, mitochondria, or microsomes. [3H]Oxytocin, added directly to particles sedimenting between 1,000 and 20,000 x g, was bound with an apparent Kd of 7.6 x 10-10 m under optimal conditions; 0.28 pmole of oxytocin was bound per mg of protein. Binding was maximal in 1 hour at 20°. The affinities of synthetic analogues of oxytocin, [4-threonine]oxytocin g oxytocin g [8-valine]oxytocin g [8-lysine]vasopressin g [4-proline]oxytocin, paralleled their milk-ejecting activities in rat mammary tissue. [3H]Oxytocin did not appear to be metabolized during incubation. Binding was enhanced by Mn2+ and Co2+ g Mg2+ g Zn2+ and was absent in the presence of 1 mm ethylenediaminetetraacetate. Ca2+, 50 mm, did not affect binding nor did 100 mm NaCl, LiCl, or KCl. Binding in 50 mm Tris-maleate buffer, containing 20 mm Mn2+, was optimum at pH 7.6. Trypsin depressed binding about 30%; phospholipases A, C, and D and neuraminidase did not. Sulfhydryl reagents such as N-ethylmaleimide and iodoacetate did not inhibit binding whereas p-hydroxymercuribenzoate did. Butanedione and sodium fluoride did not affect binding. Binding was markedly depressed by 1 mm ATP but not by guanosine triphosphate, cytosine triphosphate, hypoxanthine triphosphate, or uridine triphosphate. The binding material in the 20,000 x g particles of mammary tissue presumably is part of the oxytocin-receptor system because binding specificity paralleled biological specificity, and the high affinity of the binding sites for oxytocin is commensurate with physiological levels of the hormone. The receptor must be a protein, at least in part, because of its susceptibility to trypsin and p-hydroxymercuribenzoate.