Comparative characterization of the two primary pumps, H⁺‐ATPase and Na⁺‐ATPase, in the plasma membrane of the marine alga <i>Tetraselmis viridis</i>

Abstract

The transport characteristics of the plasma membrane H + ‐ATPase (PMHA) and Na + ‐ATPase (PMNA) from marine unicellular green alga Tetraselmis viridis Rouch. were studied using sealed plasma membrane vesicles isolated from this species. The activities of the ATPases were investigated by monitoring the ATP‐dependent pH changes in the vesicle lumen. PMHA operation led to acidification of the vesicle lumen, whereas Na + translocation into plasma membrane vesicles catalysed by PMNA was accompanied by H + efflux, namely the alkalization of the vesicle lumen (Balnokin et al., FEBS Lett 462: 402–406, 1999). The intravesicular acidification and alkalization were detected with the ΔpH probe acridine orange and the pH probe pyranine, respectively. PMHA and PMNA were found to operate in distinct pH regions, maximal activity of PMHA being observed at pH 6.5 and that of PMNA at pH 7.8. Kinetic studies revealed that the ATPases have similar affinities to their primary substrate, MgATP complex (an apparent K m = 34 ± 6.2 µ M for PMHA and 73 ± 8.7 µ M for PMNA). At the same time, the ATPases were differently affected by free Mg 2+ and ATP. Free Mg 2+ appeared to be a mixed‐type inhibitor for PMNA ( K i ′ = 210 µ M ) but it did not suppress PMHA. Conversely, free ATP markedly suppressed PMHA being a mixed‐type inhibitor ( K i ′ = 330 µ M ), but PMNA was affected by free ATP only slightly. Furthermore, the ATPases substantially differed in their sensitivities to the inhibitors of membrane ATPases, such as orthovanadate, N ‐ethylmaleimide and N , N ′‐dicyclohexylcarbodiimide. The differences found in the properties of the PMHA and PMNA are discussed in terms of regulation of their activities and their capacity to be involved in cytosolic ion homeostasis in T. viridis cells.