A plasma membrane-enriched preparation from giant barnacle muscle fibers

Abstract

We describe a procedure for obtaining a highly enriched plasma membrane ( sarcolemmal) preparation from muscle fibers of the giant barnacle ( Balanus nubilus). The sarcolemmalenriched portion migrated as a light fraction (F1) at the 10–24% sucrose interface. This fraction displayed saturable ouabain binding ( K d = 0.119 μ m) that was enriched 10 times compared to that in the original homogenate. F1 was also prepared using muscle fibers previously labeled with 1,2-ditritio-1,2(2,2′-disulfo-4,4′-diisothiocyano)diphenylethane, disodium salt ([ 3H]-H 2DIDS). F1 was enriched 25-fold in [ 3H]H 2DIDS binding sites with respect to the homogenate. Ca 2+-ATPase and succinic dehydrogenase-activities were low in F1, as was oxalate-supported Ca 2+ uptake. Compared to membranes of sarcoplasmic reticulum origin, F1 was enriched in sarcolemma membranes by about 45-fold while it was enriched approximately 30-fold over mitochondrial membranes. Thus, F1 provides an extremely pure source of external muscle membranes.