title: Purification and biochemical characterization of the F1-ATPase from Acidithiobacillus ferrooxidans NASF-1 and analysis of the atp operon.
abstract: ATPase was purified 51-fold from a chemoautotrophic, obligately acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans NASF-1. The purified ATPase showed the typical subunit pattern of the F1-ATPase on a polyacrylamide gel containing sodium dodecyl sulfate, with 5 subunits of apparent molecular masses of 55, 50, 33, 20, and 18 kDa. The enzyme hydrolyzed ATP, GTP, and ITP, but neither UTP nor ADP. The K(m) value for ATP was 1.8 mM. ATPase activity was optimum at pH 8.5 at 45 degrees C, and was activated by sulfite. Azide strongly inhibited the enzyme activity, whereas the enzyme was relatively resistant to vanadate, nitrate, and N,N'-dicyclohexylcarbodiimide. The genes encoding the subunits for the F1F(O)-ATPase from A. ferrooxidans NASF-1 were cloned as three overlapping fragments by PCR cloning and sequenced. The molecular masses of the alpha, beta, gamma, delta, and epsilon subunits of the F1 portion were deduced from the amino acid sequences to be 55.5, 50.5, 33.1, 19.2, and 15.1 kDa, respectively.
authors.affiliation: Department of Botany and Microbiology, Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Wakai
authors.forename: Asami
authors.initials: A
authors.lastname: Ohmori
authors.forename: Tadayoshi
authors.initials: T
authors.lastname: Kanao
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Sugio
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kamimura
mesh_terms: D042763:Acidithiobacillus / Q000201:enzymology*; D000255:Adenosine Triphosphate / Q000235:genetics*; D001386:Azides / Q000494:pharmacology; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009876:Operon; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000302:isolation & purification*; D013379:Substrate Specificity; D013447:Sulfites / Q000494:pharmacology; D013696:Temperature
chemical_list: D001386:Azides; D021122:Protein Subunits; D013447:Sulfites; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases