Purification of Membrane-bound ATPase of a Psychrophilic Marine Bacterium, Vibrio sp. Strain ABE-1 and Characterization as a F₀F₁-Type Enzyme

Abstract

The ATPase bound to the inner membrane of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1 (Vibrio ABE-1) was extracted with Triton X-100 and purified by fractionation with polyethylene glycol, sucrose density gradient centrifugation, and DEAE-Toyopearl 650M column chromatography. The molecular masses of subunits constituting the purified ATPase were estimated as 54, 49, 33.5, 27, 23.5, 18.5, and 15 kDa by SDS-PAGE. The composition and molecular masses of the subunits of the purified ATPase were similar to those of Escherichia coli F₀F₁-ATPase (EF₀F₁). The 54-, 49-, and 18.5-kDa polypeptides of the Vibrio ABE-1 ATPase strongly cross-reacted with the antibodies against the EF₀F₁ α, β, and b subunits, respectively. However, the Vibrio ABE-1 ATPase contained no cross-reactive polypeptide with the antibodies against A and B subunits of V-type H⁺-ATPase from mung bean tonoplasts. The ATPase activity showed two pH optimum peaks at pH 5.3 and 8.0 and was strongly inhibited by N, N'-dicyclohexyl carbodiimide (DCCD) and NaN₃. It hydrolyzed ATP, GTP, and ITP at similar rates. These properties confirm that the purified ATPase is a F₀F₁-type. The optimum temperature for the ATP-hydrolyzing activity of the enzyme was observed at 50°C, but the DCCD-sensitivity of the enzyme was markedly decreased above 30°C, suggesting that the F1-moiety is released from the enzyme complex at high temperatures. This characteristic is compatible with the psychrophilic nature of Vibrio ABE-1.