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Journal of Bacteriology, August 2003, p. 4442-4449, Vol. 185, No. 15
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.15.4442-4449.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Purification and Biochemical Characterization of the F₁F_o-ATP Synthase from Thermoalkaliphilic Bacillus sp. Strain TA2.A1

Gregory M. Cook,^1* Stefanie Keis,¹ Hugh W. Morgan,² Christoph von Ballmoos,³ Ulrich Matthey,³ Georg Kaim,³ and Peter Dimroth³

Department of Microbiology, Otago School of Medical Sciences, University of Otago, Dunedin,¹ Thermophile Research Unit, University of Waikato, Hamilton, New Zealand,² Institut für Mikrobiologie, Eidgenössische Technische Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland³

Received 5 February 2003/ Accepted 1 May 2003

We describe here purification and biochemical characterization of the F₁F_o-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F₁F_o-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F₁ subunits , ß, , , and 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 F₁F_o holoenzyme or the isolated F₁ moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F₁. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (). A transmembrane proton gradient or sodium ion gradient in the absence of 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.

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* Corresponding author. Mailing address: Department of Microbiology, Otago School of Medical Sciences, University of Otago, P.O. Box 56, Dunedin, New Zealand. Phone: 64 3 479 7722. Fax: 64 3 479 8540. E-mail: greg.cook{at}stonebow.otago.ac.nz.

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Journal of Bacteriology, August 2003, p. 4442-4449, Vol. 185, No. 15
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.15.4442-4449.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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