Purification and characterization of the catalytic moiety of vacuolar-type Na(+)-ATPase from Enterococcus hirae.
We have previously reported the molecular cloning and sequences of the ntp genes for Enterococcus hirae Na(+)-translocating ATPase [Takase, K., Kakinuma, S., Yamato, I., Konishi, K., Igarashi, K., and Kakinuma, Y. (1994) J. Biol. Chem. 269, 11037-11044]; the expected structure of this enzyme complex resembles those of the vacuolar H(+)-ATPase complexes in eukaryotes. In this paper we report purification and characterization of the catalytic moiety of Na(+)-ATPase, whose molecular size was about 400 kDa, consisting of polypeptides of 69 kDa (NtpA), 52 kDa (NtpB), and 29 kDa (NtpD) with a probable stoichiometry of 3:3:1. Purified enzyme hydrolyzed GTP as the best substrate (GTP > CTP > UTP > ATP), and the activity was maximal at around pH 6.0. The activity was not stimulated by sodium ions, and was selectively inhibited by nitrate. These properties were different from those of membrane-bound Na(+)-ATPase, suggesting that a significant conformational change of the catalytic moiety may take place upon dissociation from the membrane-embedded moiety and probably also loss of other hydrophilic subunits. Antiserum against purified enzyme inhibited the Na(+)-stimulated ATPase activity of the membranes. Immunoblotting analysis revealed that the change in the amounts of A and B subunits of the membranes paralleled that of the Na(+)-ATPase activity. Furthermore, the A subunit was missing in the membranes of a Na(+)-ATPase mutant, and recovered in those of its revertant. These immunochemical data are consistent with the notion that this enzyme is the hydrophilic catalytic moiety of the V-type Na(+)-ATPase in E. hirae.
Kakinuma
Y
Y
Faculty of Pharmaceutical Sciences, Chiba University.
Igarashi
K
K
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D027682:Cation Transport Proteins; D016983:Enterococcus / Q000201:enzymology*; D007150:Immunohistochemistry; D014617:Vacuoles / Q000201:enzymology*
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase