Purification of membrane-bound ATPase of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1 and characterization as a F0F1-type enzyme.
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 alpha, beta, 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 degrees C, but the DCCD-sensitivity of the enzyme was markedly decreased above 30 degrees 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.
Harashima
A
A
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Japan.
Takada
Y
Y
Fukunaga
N
N
D003429:Cross Reactions; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D013379:Substrate Specificity; D013696:Temperature; D014733:Vibrio / Q000201:enzymology* / Q000648:ultrastructure
D008565:Membrane Proteins; D010446:Peptide Fragments; D006180:Proton-Translocating ATPases