Construction, expression and characterization of a plasmid-encoded Na(+)-specific ATPase hybrid consisting of Propionigenium modestum F0-ATPase and Escherichia coli F1-ATPase.
The Escherichia coli strain DK8, a deletion mutant lacking the complete unc operon, was transformed with a plasmid containing the genes encoding the a, b, c, delta and part of the alpha subunit of the Na(+)-dependent ATPase of Propionigenium modestum and the genes encoding the alpha, gamma, beta and epsilon subunits of the H(+)-dependent E. coli ATPase. The transformants showed Na(+)-dependent growth on succinate as non-fermentable carbon source. The functionally expressed hybrid ATPase was activated 13-fold at pH 7.5 by the addition of Na+ and inhibited by 1,3-dicyclohexylcarbodiimide, azide and tributyltin chloride. At pH 7.5 and pH 9.0, the hybrid enzyme was protected from inhibition by 1,3-dicyclohexylcarbodiimide in the presence of 50 mM NaCl and 5 mM NaCl, respectively. The hybrid ATPase was reconstituted into proteoliposomes and catalyzed the transport of Na+ upon ATP addition. ATP-dependent fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine proved that the ATPase hybrid was able to pump protons in the absence of Na+. Furthermore, ATP synthesis could be measured under conditions where a valinomycin-mediated K+ diffusion potential (delta psi) and a Na+ concentration gradient (delta p Na+) were imposed.
Kaim
G
G
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Dimroth
P
P
D000251:Adenosine Triphosphatases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics; D027682:Cation Transport Proteins; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D007700:Kinetics; D009876:Operon; D010957:Plasmids; D055503:Protein Multimerization; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D012964:Sodium / Q000494:pharmacology; D014169:Transformation, Bacterial
D027682:Cation Transport Proteins; D000255:Adenosine Triphosphate; D012964:Sodium; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase; D006180:Proton-Translocating ATPases