Isolation of a Plasma‐Membrane Subfraction from Rat Liver Containing an Insulin‐Sensitive Cyclic‐AMP Phosphodiesterase

作者：P.D.R. House, Parissa Poulis, Maurice J. Weidemann · 发表于：European Journal of Biochemistry · 年份：1972 · DOI：10.1111/j.1432-1033.1972.tb19703.x · 被引用次数：112 · 研究领域：Pancreatic function and diabetes、Ion Transport and Channel Regulation、Adenosine and Purinergic Signaling

A procedure for the isolation and separation of three different subfractions of rat liver plasma membranes is described. The subfractions have different enzyme activities and sucrose buoyant densities of 1.18, 1.16 and 1.13 g/ml respectively. It was found that the low‐density subfraction had the following properties: It appeared as smooth membrane vesicles approximately 0.1–0.2 μm in diameter when visualized under positive and negative staining with the electron microscope. Its contamination by microsomes on a protein basis was less than 3% as measured by glucose‐6‐phosphatase activity, and no succinic dehydrogenase activity or DNA were detected. The low‐density subfraction contained high relative specific activities of 5′‐nucleotidase, (Na+, K+)‐Mg2+‐stimulated ATPase, phosphodiesterase, and a glucagon and adrenalin‐sensitive adenylate cyclase. This fraction yielded over 30% of the total 5′‐nucleotidase, adenylate cyclase and phosphodiesterase activities of the homogenate. A specific 3′: 5′‐AMP phosphodiesterase activity of this subfraction was stimulated by insulin. The half‐maximal concentration required to produce this stimulation was 2.3 nM. The stimulation was already observed at the shortest time interval tested (30 sec). The localization in situ of the low‐density membrane subfraction on the surface of the hepatic cell is discussed, and it is suggested that it is derived mainly from the microvillar surface of hepatic parenchymal cells facing the perisinusoidal space.