The two major ATPase activities of intact and leaky cardiac membrane vesicles (microsomes) were characterized with respect to ionic activation requirements. The predominant ATPase activity of intact vesicles was (K+ + Ca2+)-ATPase, an enzymic activity localized to sarcoplasmic reticulum, whereas the predominant ATPase activity of leaky, sodium dodecyl sulfate-pretreated vesicles was (Na+ + K+)-ATPase, an enzymic activity localized to sarcolemma. The (K+ + Ca2+)-ATPase activity was stimulated 4- to 5-fold by 100 mM K+ in the presence of 50 μM Ca2+. Phosphorylation of the (K+ + Ca2+)-ATPase of intact vesicles with [γ-32P]ATP was Ca2+ dependent, and monovalent cations including K+ increased the level of [32P]phosphoprotein by up to 50% when phosphorylation was measured at 5°C. After the intact vesicles were treated with SDS (0.30 mg/ml), (K+ + Ca2+)-ATPase was inactivated, as was Ca2+-dependent 32P incorporation. The monovalent cation-stimulated ATPase activity of the particulate residue (SDS-extracted membrane vesicles) displayed the usual characteristics of ouabain-sensitive (Na+ + K+)-ATPase and the activity was increased 9- to 14-fold over the small amount of patent (Na+ + K+)-ATPase activity of intact membrane vesicles. 32P incorporation by the (Na+ + K+)-ATPase of SDS-extracted vesicles was Na+ dependent, and Na+-stimulated incorporation was increased 7- to 9-fold over that of intact vesicles. Slab gel polyacrylamide electrophoresis of both intact and SDS-extracted crude vesicle preparations revealed at least 40 distinct Coomassie Blue-positive protein bands and provided evidence for a possible heterogeneous membrane origin of the vesicles. Periodic acid-Schiff staining of the gels revealed at least two major glycoproteins. Simultaneous electrophoresis of the 32P-intermediates of the (K+ + Ca2+)-ATPase and the (Na+ + K+)-ATPase in the same gels did not resolve the two enzymes clearly. With sucrose gradient centrifugation of intact membrane vesicles, it was possible to physically resolve the two ATPase activities. Latent (Na+ + K+)-ATPase activity (unmasked by exposing the various fractions to SDS) was found in the higher regions of the gradient, whereas (K+ + Ca2+)-ATPase activity was primarily in the denser regions. A reasonable interpretation of the data is that cardiac microsomes consist of membrane vesicles derived both from sarcolemma and sarcoplasmic reticulum. (Na+ + K+)-ATPase is localized to intact vesicles of sarcolemma but is mainly latent, whereas (K+ + Ca2+)-ATPase is mostly patent and is localized to vesicles of sarcoplasmic reticulum.
ASJC Scopus subject areas
- Cell Biology