A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na+-dependent H+ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na+ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with 22Na+ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na+-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent 22Na+ uptake and abolished the electric potential generated by the Na+-ATPase at the vesicle membrane. A permeant anion NO− 3 accelerated ATP-dependent 22Na+ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO− 3 inhibited the ATP-and Na+-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na+-dependent H+ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na+-ATPase. Hence, the Na+-transporting ATPase of D. maritima carries only one ion species, i.e., Na+. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.
Russian Journal of Plant Physiology – Springer Journals
Published: Jul 7, 2006
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