Purification and reconstitution of Na+-translocating vacuolar ATPase from Enterococcus hirae.
Vacuolar ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. An unusual member of this family, a sodium-translocating ATPase, has been found in the eubacterium Enterococcus hirae. We report here the purification of enterococcal Na+-ATPase from the plasma membrane of cells, whose ATPase content was highly amplified by expression of the cloned ntp operon that encodes this Na+-ATPase (ntpFIKECGABDHJ). The purified enzyme appears to consist of nine Ntp polypeptides, all the above except for the ntpH and ntpJ gene products. ATPase activity was strictly dependent on the presence of Na+ or Li+ ions and was inhibited by nitrate, N-ethylmaleimide, and the peptide antibiotic destruxin B. When the purified ATPase was reconstituted into liposomes prepared from Enterococcus faecalis phospholipids, ATP-driven Na+ uptake was observed; uptake was blocked by nitrate, destruxin B, and monensin, but it accelerated by carbonyl cyanide m-chlorophenylhydrazone and valinomycin. These data demonstrate that E. hirae Na+-ATPase is an electrogenic sodium pump of the vacuolar type. This is a promising system for research on the fundamental molecular structure and mechanism of vacuolar ATPase.
Murata
T
T
Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi, Chiba 278, Japan.
Takase
K
K
Yamato
I
I
Igarashi
K
K
Kakinuma
Y
Y
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000906:Antibodies; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D016983:Enterococcus / Q000201:enzymology*; D013293:Enterococcus faecalis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005798:Genes, Bacterial; D007700:Kinetics; D008081:Liposomes; D008094:Lithium / Q000494:pharmacology; D009876:Operon; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011510:Proteolipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D013104:Spheroplasts / Q000201:enzymology
D000906:Antibodies; D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D008081:Liposomes; D010446:Peptide Fragments; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D008094:Lithium; D012964:Sodium; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase