Na(+)-stimulated ATPase of alkaliphilic halotolerant cyanobacterium Aphanothece halophytica translocates Na(+ )into proteoliposomes via Na(+ )uniport mechanism

BACKGROUND: When cells are exposed to high salinity conditions, they develop a mechanism to extrude excess Na(+ )from cells to maintain the cytoplasmic Na(+ )concentration. Until now, the ATPase involved in Na(+ )transport in cyanobacteria has not been characterized. Here, the characterization of ATPase and its role in Na(+ )transport of alkaliphilic halotolerant Aphanothece halophytica were investigated to understand the survival mechanism of A. halophytica under high salinity conditions. RESULTS: The purified enzyme catalyzed the hydrolysis of ATP in the presence of Na(+ )but not K(+), Li(+ )and Ca(2+). The apparent K(m )values for Na(+ )and ATP were 2.0 and 1.2 mM, respectively. The enzyme is likely the F(1)F(0)-ATPase based on the usual subunit pattern and the protection against N,N'-dicyclohexylcarbodiimide inhibition of ATPase activity by Na(+ )in a pH-dependent manner. Proteoliposomes reconstituted with the purified enzyme could take up Na(+ )upon the addition of ATP. The apparent K(m )values for this uptake were 3.3 and 0.5 mM for Na(+ )and ATP, respectively. The mechanism of Na(+ )transport mediated by Na(+)-stimulated ATPase in A. halophytica was revealed. Using acridine orange as a probe, alkalization of the lumen of proteoliposomes reconstituted with Na(+)-stimulated ATPase was observed upon the addition of ATP with Na(+ )but not with K(+), Li(+ )and Ca(2+). The Na(+)- and ATP-dependent alkalization of the proteoliposome lumen was stimulated by carbonyl cyanide m - chlorophenylhydrazone (CCCP) but was inhibited by a permeant anion nitrate. The proteoliposomes showed both ATPase activity and ATP-dependent Na(+ )uptake activity. The uptake of Na(+ )was enhanced by CCCP and nitrate. On the other hand, both CCCP and nitrate were shown to dissipate the preformed electric potential generated by Na(+)-stimulated ATPase of the proteoliposomes. CONCLUSION: The data demonstrate that Na(+)-stimulated ATPase from A. halophytica, a likely member of F-type ATPase, functions as an electrogenic Na(+ )pump which transports only Na(+ )upon hydrolysis of ATP. A secondary event, Na(+)- and ATP-dependent H(+ )efflux from proteoliposomes, is driven by the electric potential generated by Na(+)-stimulated ATPase.