The functional unit of calcium-plus-magnesium-ion-dependent adenosine triphosphatase from sarcoplasmic reticulum. The aggregational state of the deoxycholate-solubilized protein in an enzymically active form.

Vesicles consisting of (Ca(2+)+Mg(2+))-dependent ATPase (adenosine triphosphatase), and lipid were prepared from sarcoplasmic reticulum of rabbit skeletal muscle. As with non-ionic detergents [le Maire, Møller & Tanford (1976) Biochemistry15, 2336-2342] the (Ca(2+)+Mg(2+))-dependent ATPase after solubilization by deoxycholate showed a pronounced tendency to form oligomers in gel-chromatographic experiments, when eluted in the presence of deoxycholate and phosphatidylcholine. To evaluate the functional significance of oligomer formation the properties of enzymically active preparations of ATPase, solubilized by deoxycholate, were studied. Such preparations were obtained at a protein concentration of 2.5mg/ml in the presence of a high salt concentration (0.4m-KCl) and sucrose (0.3m) in the solubilization medium. Analytical ultracentrifugation of solubilized ATPase showed one protein boundary moving at the same rate as gel-chromatographically prepared monomeric ATPase (s(20,w)=6.0S). From simultaneous measurements of the diffusion coefficient an apparent molecular weight of 133000 was calculated, consistent with solubilization of ATPase in predominantly monomeric form. The enzymic activity of deoxycholate-solubilized ATPase when measured directly in the solubilization medium at optimal Ca(2+) and MgATP concentrations was about 35-50% of that of vesicular ATPase. The dependence of enzymic activity on MgATP concentration indicated that the solubilized ATPase retained high-affinity binding of MgATP, but the presence of high concentrations of the nucleotide did not stimulate activity further, in contrast with that of vesicular ATPase. The dependence of enzymic activity on the free Ca(2+) concentration was essentially the same for both solubilized and vesicular forms, indicating that interaction of ATPase with more than one molecule of Ca(2+) is required for enzyme activity. Solubilized enzyme at 20 degrees C was phosphorylated to about the same degree as vesicular ATPase. It is concluded that the catalytic activity of monomeric ATPase retains most of the features of vesicular ATPase and that extensive oligomer formation in gel-chromatographic experiments in the presence of deoxycholate probably reflects processes taking place during inactivation and delipidation of the protein.