Presence of a Na+-stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium Aphanothece halophytica

Aphanothece cells could take up Na+ and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na+ exhibited Na+ extrusion ability upon energizing with glucose. Na+ was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na+-ionophore. Orthovanadate and CCCP strongly inhibited Na+ uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na+ but not with K+, Ca2+ and Li+. The K-m values for ATP and Na+ were 1.66 +/- 0.12 and 25.0 +/- 1.8 mM, respectively, whereas the V-max value was 0.66 +/- 0.05 mu mol min(-1) mg(-1). Mg2+ was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na+/H+ antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na+-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.