A plasma membrane-enriched preparation from giant barnacle muscle fibers

We describe a procedure for obtaining a highly enriched plasma membrane (sarcolemmal) preparation from muscle fibers of the giant barnacle (Balanus nubilus). The sarcolemmal-enriched portion migrated as a light fraction (F1) at the 10-24% sucrose interface. This fraction displayed saturable ouabain binding (Kd = 0.119 microM) 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)diphenylet hane, disodium salt [( 3H]-H2DIDS). F1 was enriched 25-fold in [3H]H2DIDS binding sites with respect to the homogenate. Ca2+-ATPase and succinic dehydrogenase-activities were low in F1, as was oxalate-supported Ca2+ 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.