A protein component which accounts for lOO/, of the total protein of isolated cell membranes of Micrococcus lysodeikticus has been partially characterized. The protein can be released into solution from the membrane complex and in this "soluble" state it possesses Ca++-dependent adenosine triphosphatase activity. This enzyme has been purified about 50-fold, by a procedure based on its association-dissociation properties with the membrane. The progressive purification and degree of homogeneity have been demonstrated by examining the various stages in the soluble ATPase preparation by disc electrophoresis and by gel diffusion against membrane antiserum. On treatment with 8 M urea or 1 O/, sodium dodecyl sulfate followed by disc electrophoresis, clear evidence of dissociation was obtained, indicating the existence of subunits. The purified protein has a sedimentation coefficient of 14-15 S as determined by sucrose gradient centrifugation or analytical ultracentrifugation. A sedimentation coefficient of 3.5 S has been estimated for the main product of the protein dissociated by sodium dodecyl sulfate again an indication of the dissociation of the protein into subunits, probably identical on the basis of molecular weight. The "soluble" enzyme was activated by Ca++, optimum activation being observed a t ATP/Ca++ ratio of 131 and pH 7.5 under which conditions 50-60 pmoles of ATP were hydrolyzed per mg protein per 10 min at 37". The ATPase was quite stable a t 4". The Ca++-dependent ATPase activity was inhibited by ADP, sodium azide, and Mg++.When the membrane-ATPase complex remains stable, no hydrolytic activity for ATP is shown unless trypsin activation. Our system shows similarities with the "allotopic" properties described for some mitochondrial enzymes. From this and previous work in our laboratory a close relationship between the "stalked particles" seen in M . lysodeikticus cell membranes by electron microscopy and ATPase appears to be evident.
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