Characterization of Na+- atpase in the halotolerant cyanobacterium aphanothece halophytica

Abstract

Extrusion of sodium ion is needed to balance and maintain sodium level for microorganisms to survive in hypersalin environment. Activity of Na+- ATPase in halotolerant cyanobacterium aphanothece haolphytica was investigated. Cells were preloaded with 22Na+ and the extrusion of 22Na+ from cells was followed by energizing the cells with glucose. To ascertain that this Na+ extrusion is Na+- ATPase, the plasma membranes vesicles were prepared by aqueous polymer two-phase partitioning with dextran and polyethylene glycol and used for the assay of Na+- ATPase activity by measuring ATP hydrolysis. The ATPase activities were stimulated to greatest extent in the presence of 100 mM Na+ and 5 mM Mg2+ ions, pH 7.6. The K[subscript m] value for ATP and Na+ ions were 1.66 mM and 25 mM respectively. The maximum velocities (V [subscript max])were 0.66 and 0.50 umol/min/mg protein using ATP and Na+ as variable substrate respectively. The activity was inhibited by vanadate, a potent inhibitor of P-type ATPase. The activity was decreased about half of original value by 0.01 mM socium vanadate and 0.20 mM gramicidin. Alternatively, the membrane vesicle containing Na+- ATPase, exhibited 22Na+ transport by the addition of ATP, which was inhibited by sodium ionophore and gramicidin. It is likely that the Na+- ATPase belongs to P-type and is involved in Na+ transport. The enzyme activity was enhanced by the high salinity of growth medium.