Intact Vesicles of Canine Cardiac Sarcolemma

Most biological membranes are functionally asymmetric. To study biochemical control of cardiac transsarcolemmal ion fluxes, it would be of obvious advantage to use isolated vesicles of sarcolemma which retains the low passive permeability characteristics of intact sarcolemma because in such vesicles the membrane should exhibit its normal asymmetric character with respect to enzymic activities. The purpose of this investigation was to attempt to identify such vesicles in a cardiac microsomal (membrane vesicular) preparation. We studied activation by Na+ and K of Na+,KATPase and its associated K-phosphatase activities, using as substrates ATP or p-nitrophenytphosphate (pNPP) in the presence of Mg. Optimal concentrations of K alone (10 mM) stimulated p-nitrophenylphosphatase (pNPPase) activity 1.8-fold, and over 80% of the increase could be inhibited by ouabain. Optimal Na plus K' concentrations (100 mM and 10 DIM, respectively) stimulated the rate of ATP hydrolysis 2-fold, but only 11 ± 1.1% of the increased activity was ouabain-sensitive. Optimal pretreatment with sodium dodecyl sulfate (SDS) (0.3 mg/ml) rendered both activities completely sensitive to inhibition by ouabain and reduced the basal Mg-ATPase activity by 70-90%. The K-stimulated pNPPase activity doubled after preincubation in SDS, but the ATPase activity stimulated by Na+ plus K fell by 50% under these conditions. A similar pattern of apparent activation was produced by preincubation with deoxycholate (DOC), except that basal Mg-dependent activities were resistant to destruction by this detergent. The incremental responses to activation by ions and substrates, and inhibition by ouabain, are consistent with the hypothesis that permeability-intact vesides of sarcolemma are present in the isolated preparation, and that detergent activation renders the vesides highly permeable to the ions, substrates, and ouabain.