Cardiac adenylate cyclase. I. Preparation and characterisation of a subcellular fraction containing catecholamine-sensitive adenylate cyclase

Abstract

Differential centrifugation of homogenates of rat ventricles yielded subcellular fractions which were assayed for adenylate cyclase activity, as well as enzymes associated predominantly with certain intracellular structures. The distribution of adenylate cyclase activity paralleled only that of the marker enzyme for sarcolemma, 5′-AMPase. However, the heterogeneity of the particulate fractions made it impossible to ascertain the exact intracellular localization of adenylate cyclase. Histological examination of the fraction (P 1) containing the bulk of the adenylate cyclase activity revealed large numbers of isolated myofibres. Purification of this fraction was effected by homogenizing it in a large volume of isotonic sucrose to disperse and dissolve the myofibrillar matrix. The resultant homogenate was then subjected to differential centrifugation. A pellet (P 4) was obtained containing only slight activity of marker enzymes for mitochondria and sarcoplasmic reticulum, but which was enriched in 5′-AMPase activity, plasma membranes (as revealed by its cholesterol/phospholipid ratio) and possessed a reduced muscle protein content. Histological examination of this fraction revealed an absence of myofibres. The specific activity of the basal, isoprenaline—and fluoride—stimulated adeynlate cyclase in P 4 was doubled relative to P 1 and this fraction contained approximately half of the total adeynlate cyclase activity of P 1. It was concluded that the bulk of the catecholamine-sensitive adenylate cyclase activity of the cardiac muscle homogenate was localized in the sarcolemma. The described procedure for the isolation of a purified sarcolemma fraction is rapid and does not require the use of an ultracentrifuge or density gradients.