Membrane ATPase of Bacillus subtilis. I. Purification and properties.

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step. The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s20,w of 13,4 and an amino acid composition very similar to bacterial ATPases already studied. After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (alpha) and 57 000 (beta) with different charges. Kinetic studies showed that Ca2+ or Zn2+ are required for ATPase activity, although Mg2+ was uneffective. At optimal Ca2+ concentration, the Mg2+ has an inhibitory effect. The Km for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na+ + K+)-ATPase are studied.