title: Purification and biochemical characterization of the F1Fo-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1.
abstract: We describe here purification and biochemical characterization of the F(1)F(o)-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F(1)F(o)-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F(1) subunits alpha, beta, gamma, delta, and epsilon and F(o) subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F(1)F(o) holoenzyme or the isolated F(1) moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F(1). After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Deltapsi). A transmembrane proton gradient or sodium ion gradient in the absence of Deltapsi was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na(+)/H(+) antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na(+) ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.
authors.affiliation: Department of Microbiology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand. greg.cook@stonebow.otago.ac.nz
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Cook
authors.forename: Stefanie
authors.initials: S
authors.lastname: Keis
authors.forename: Hugh W
authors.initials: HW
authors.lastname: Morgan
authors.forename: Christoph
authors.initials: C
authors.lastname: von Ballmoos
authors.forename: Ulrich
authors.initials: U
authors.lastname: Matthey
authors.forename: Georg
authors.initials: G
authors.lastname: Kaim
authors.forename: Peter
authors.initials: P
authors.lastname: Dimroth
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011510:Proteolipids / Q000378:metabolism; D020539:Sequence Analysis, Protein
chemical_list: D011510:Proteolipids; C020485:proteoliposomes; D000255:Adenosine Triphosphate; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases