Preparation of rat liver plasma membrane with respect to glucagon-sensitive adenylate cyclase

Abstract

Further modification of Neville's method of preparation of rat liver plasma membrane has been made in order to study glucagon-sensitive adenylate cyclase. This modified method introduces two additional steps to the Neville procedures. One involves saving an intermediate layer above the pellet following 1500g centrifugation. The other adds a centrifugation step at 20,000g. The improved method increases the yield of membrane protein by 5-fold and increases forskolin, glucagon, and 5′-guanyl imidodiphosphate stimulated activity of adenylate cyclase by 2.8-fold. A 13-fold increase in the yield of total adenylate cyclase activity above the current method was obtained. The membrane adenylate cyclase and its hormone sensitivity was stable in liquid nitrogen for at least 8 months. This modified method appears to be useful in preparing a better yield and quality of rat liver plasma membrane from given starting hepatic tissue for studies of glucagon-sensitive adenylate cyclase.