The Purification of Bovine Thyroid Plasma Membranes and the Properties of Membrane-bound Adenyl Cyclase

Abstract

Abstract Bovine thyroid membranes have been purified about 100-fold on the basis of thyrotropin- or F--stimulated adenyl cyclase activity. These activities copurified with Na+-K+-requiring adenosine triphosphatase, K+-stimulated p-nitrophenyl phosphatase, and 5'-nucleotidase. The adenyl cyclase of these preparations is stimulated over a wide range of bovine thyrotropin concentrations, by human chorionic thyrotropin, and sometimes by prostaglandin E1, but not by long acting thyroid stimulator. The adenyl cyclase is stable in the frozen state but loses thyrotropin and F- responsiveness rapidly at 43°. Maximal activity is obtained with a Mg++:ATP ratio of 2 and when the ATP concentration exceeds 2 mm. The thyrotropin response is enhanced by K+ ion whereas the F- response is not affected. Li+ ion inhibits strongly, especially when the Mg++ concentration is limiting. Na+ is weakly inhibitory. Both F-- and thyrotropin-stimulated cyclase activity are strongly inhibited by N-ethylmaleimide, p-chlormercuribenzoate, sodium dodecyl sulfate, filipin, sodium tetraphenylboron, valinomycin, and monensin A. Another class of agents, which includes the phenothiazines, thymol, cobramine B, and gramicidin S, inhibits thyrotropin stimulation of the enzyme but enhances the F- response. It is concluded that the thyrotropin and the adenyl cyclase (F- activation) are distinct entities, and it is suggested that prostaglandin E1 uses a different receptor than thyrotropin.