Purification of Bovine Adrenal‐Cortex Plasma‐Membrane Vesicles Containing a Highly Corticotropin‐Sensitive Adenylate‐Cyclase System and Angiotensin‐II‐Binding Sites

Abstract

The purpose of this experimental investigation was to provide a purified plasma membrane fraction containing a highly hormone-responsive adenylate cyclase [AC] system. Bovine adrenal cortex was homogenized and a washed pellet (450,000 .times. g .cntdot. min) was fractionated by zonal centrifugation in a sucrose and dextran gradient. AC activity was purified up to 60-fold to a specific activity of 55, 340 and 210 pmol of cyclic AMP produced per minute per mg of protein at 38.degree. C for the basal, ACTH and F--activated states, respectively. The time course of the AC activity is linear. The concentration necessary for half-maximal stimulation by ACTH-(1-24)-tetracosipeptide is 0.5 .mu.M. The high hormone-responsiveness of the membrane preparation allows one to demonstrate activation of AC by very weakly agonistic ACTH fragments. The F--activated state can be detergent-dispersed by Lubrol and shows a Km (ATP) different from that of either the basal or ACTH-stimulated state. Other marker enzymes such as 5''-nucleotidase, glucose-6-phosphatase and cytochrome oxidase were followed during purification. The plasma membrane fraction shows rather homogeneous, relatively large vesicles (mean diameter 0.5 .mu.m). It contains high-affinity binding sites for angiotensin II (about 2 pmol/mg protein) with an apparent association constant of 2 .times. 107 (l/mol) at 12.degree. C. The yield, 20 mg of membrane protein per preparation, may make it a tool in either affinity-labeling studies with the peptide hormones mentioned or the starting point for solubilization and purification of AC.