Characterization of the H(+)-pumping F1F0 ATPase of Vibrio alginolyticus.
The F1F0 ATPase of Vibrio alginolyticus was cloned from a chromosomal lambda library. The unc operon, which contains the structural genes for the ATPase, was sequenced and shown to have a gene organization of uncIBEFHAGDC. The sequence of each subunit was compared with those of other eubacterial ATPases. The V. alginolyticus unc genes exhibited greater similarity to the Escherichia coli unc genes than to any of the other bacterial unc genes for which the sequence is available. The ATPase was expressed in an E. coli unc deletion strain, and the ATP hydrolytic activity was characterized. It has a pH optimum of 7.6 and is stimulated by the addition of Triton X-100 or any of a variety of salts. The recombinant F1F0 was purified 30.4-fold and reconstituted into proteoliposomes. This enzyme catalyzed the pumping of protons coupled to ATP hydrolysis as measured in fluorescence quenching experiments but would not pump Na+ ions under similar conditions.
Krumholz
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Department of Biological Sciences, Stanford University, California 94305.
Esser
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U
Simoni
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009876:Operon; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D006180:Proton-Translocating ATPases