Membrane Mg 2+ -(Ca 2+ )-Activated Adenosine Triphosphatase of Escherichia coli : Characterization in the Membrane-Bound and Solubilized States

Abstract

The membrane-associated Mg 2+ -activated and Ca 2+ -activated adenosine 5′-triphosphatase (EC 3.6.1.3; ATPase) activities of Escherichia coli were further characterized. The degree of inhibition of membrane-bound Mg 2+ -(Ca 2+ )-ATPase by a series of anions (i.e., sodium salts of nitrate, iodide, chloride, and acetate) was found to correlate with the relative chaotropic, or solubilizing, effectiveness of these anions. The enzyme was solubilized from washed membrane ghosts by treatment with 0.04% sodium lauryl sulfate at p H 9.0 and 37 C. Solubilized Mg 2+ -(Ca 2+ )-ATPase exhibited an initial increase in activity, followed by fairly rapid inactivation, both ATPase activities being particularly cold-labile. The combined stabilizing effects of lauryl mercaptan (1-dodecanethiol), 0.01 m tris(hydroxymethyl)amino-methane-hydrochloride buffer ( p H 9.0), 0.2 m m MgCl 2 , and ambient temperature facilitated partial purification of the enzyme, the molecular weight of which was estimated to be approximately 100,000 by the gel filtration technique. In general, the membrane-associated Mg 2+ -(Ca 2+ )-ATPase of E. coli resembles both mitochondrial membrane ATPase and the well-characterized membrane ATPases of Bacillus megaterium and Microcococcus lysodeikticus . It is of particular interest that N,N ′-dicyclohexylcarbodiimide (DCCD), a known inhibitor of mitochondrial ATPase, of mitochondrial oxidative phosphorylation, and of the membrane-bound Mg 2+ -ATPase of Streptococcus faecalis was found to inhibit both the membrane-bound and the solubilized forms of E. coli Mg 2+ -(Ca 2+ )-ATPase. The sensitivity of the membrane-associated Mg 2+ -(Ca 2+ )-ATPase of E. coli to both anions and cations, its allotopic behavior, and its susceptibility to inhibition by DCCD favor the idea that this enzyme plays a key, probably polyfunctional, role in such biological activities of the membrane as oxidative phosphorylation and ion transport.