An improved technique for preparation of skeletal muscle cell plasma membrane

An improved technique of plasma membrane isolation from rat skeletal muscle is presented, based on the cautious disruption of the muscle fibers, elimination of actomyosin by salt solutions and separation of sarcolemma from organelles by differential centrifugation.

Abstract

1. An improved technique of plasma membrane isolation from rat skeletal muscle is presented. The procedure is based on the cautious disruption of the muscle fibers, elimination of actomyosin by salt solutions and separation of sarcolemma from organelles by differential centrifugation. This is followed by a centrifugation on a discontinuous sucrose density gradient. The plasma membranes are yielded in the form of vesicles. They are characterized and their purity is assessed by electron microscopy, chemical and biochemical tests. 2. The fraction of pure plasma membrane is found to have a high specific activity in plasma membrane markers: 5′-nucleotidase, (Na+,K+)-stimulated Mg2+-dependent ouabain-sensitive ATPase and a high molar ratio of cholesterol to phospholipids; these markers have lower or negligible specific activities in the other fractions. The enzymatic tests performed to determine the cross-contamination by cell organelles are: succinate dehydrogenase and NADH:cytochrome c-reductase rotenone insensitive, for mitochondria and outer-mitochondrial membrane; NADPH: cytochrome c-reductase for sarcoplasmic reticulum; IDP phosphohydrolase for Golgi apparatus: acid phosphatase for lysosomes; and tentatively lactate-dehydrogenase for cytoplasm. These activities are poorly represented or absent in the plasma membrane fraction.