Isolation of prokaryotic V0V1-ATPase from a thermophilic eubacterium Thermus thermophilus.
The soluble ATPase purified from an aerobic thermophilic eubacterium, Thermus thermophilus, was not a usual F1-ATPase but a V1-ATPase, a peripheral section of plasma membrane V-type ATPase (Yokoyama, K., Oshima, T., and Yoshida, M. (1990) J. Biol. Chem. 265, 21946-21950). Here, we report the purification of V-type ATPase from the same bacterium (V0V1-ATPase) which consists of V1-ATPase and a membrane-integrated section, V0. The V0V1-ATPase, either in the Triton X-100-solubilized membrane fraction or in the purified state, migrates as a single band in a non-denaturing polyacrylamide gel electrophoresis for membrane protein complexes, and eight kinds of polypeptides are found when this band is developed in a second dimension denaturing gel electrophoresis in the presence of sodium dodecyl sulfate. The 66-, 56-, 30-, and 12-kDa polypeptides are the subunits of V1-ATPase and the 100-, 38-, 24-, and 13-kDa polypeptides are candidates for V0 (or V0-associated) subunits. The amino-terminal amino acid sequences of the 38- and 24-kDa subunits do not show obvious similarity to any subunits of eukaryotic V0V1-ATPases. The kinetic properties of the purified V0V1-ATPase are very similar to those of V1-ATPase: a very low ATPase activity, stimulation by bisulfite, inhibition by nitrate, and resistance against inhibitors of eukaryotic V-type ATPases, Bafilomycin A1 and N-ethylmaleimide. The V0 vesicles prepared from reconstituted V0V1-ATPase vesicles by 6 M urea treatment show the H+ channel activity. This H+ channel activity is abolished either by treatment of vesicles with dicyclohexylcarbodiimide or by the back addition of V1-ATPase. These results indicate that the coupling ion of this V0V1-ATPase is H+.
Yokoyama
K
K
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Yokohama, Japan.
Akabane
Y
Y
Ishii
N
N
Yoshida
M
M
D000595:Amino Acid Sequence; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015388:Organelles / Q000201:enzymology / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000201:enzymology*; D014634:Valinomycin / Q000494:pharmacology
D046911:Macromolecular Substances; D010446:Peptide Fragments; D014634:Valinomycin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D006180:Proton-Translocating ATPases