Isolation of plasma membrane vesicles, derived from transverse tubules, by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media

Abstract

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. (Na + + K +)-stimulated , Mg 2+-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca 2+-stimulated, Mg 2+-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell Cell segments in an amber low-speed ( 800 × g) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at 100 000 × g for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of (Na + + K +)-stimulated ATPase compared with the other fractions, but it had essentially no Ca 2+-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate. Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined by a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basement membrane, could not be detected in this fraction.