The dissipation of membrane potential would result in complete suppression of H efflux, if the membrane potential across the proteoliposome generated by Na stimulated ATPase were the only driving force for H extrusion. On the other hand, if H efflux occurred directly via the Na stimulated ATPase as an antiporter by exchanging Na for H , the dissipation of the membrane potential would not suppress H efflux . A permeant anion, nitrate, that generally penetrates easily across biological membranes down its electrochemical gradient is often used as a charge compensating anion to abolish membrane potential across proteoliposomes during the measurements of ATP driven H translocation. If nitrate inhibits the ATP dependent alkalization of the proteoliposome lumen and promotes both dissipation of membrane potential and ATP dependent Na uptake, it can be concluded that ATP and Na dependent H efflux from proteoliposomes is driven by the membrane potential generated by Na ATPase.
Hence, Na ATPase operates as a uniporter carrying only Na , and H is not involved in the catalytic cycle of this enzyme. On the contrary, if nitrate increases both ATP dependent alkalization of the proteoliposome lumen and Na uptake while membrane potential is dissipated, it can be concluded that Na stimulated ATPase operates as an antiporter catalyzing Na H exchange. Our results are consistent with mechanism 1 by which the purified Na stimulated ATPase operates as a PF-02341066 distributor uniporter. The ATP dependent Na uptake in proteoliposomes was accelerated in the presence of CCCP and nitrate and both agents dissipated membrane potential generated by Na stimulated ATPase . CCCP stimulated ATP dependent alkalization of proteoliposomes while nitrate inhibited this alkalization . Only Na but not K , Li and Ca2 promoted H efflux . The results indicate that H efflux from proteoliposomes is driven by the membrane potential generated by Na stimulated ATPase and is specific for Na . Moreover, this dependence of lumen alkalization on Na coincided with the requirement of Na but not K , Li and Ca2 on ATPase activity .
Therefore, we can conclude that 1 Na stimulated ATPase from A. halophytica operates as a uniporter which takes up only Na while H is counterion and 2 ATP dependent lumen alkalization occurred as a result of the operation of the Na stimulated ATPase. H efflux does not occur directly via Na stimulated ATPase but is driven by the membrane potential generated during Na stimulated ATPase operation. The operation of Na stimulated ATPase as a Na uniporter has been previously reported in the halotolerant y27632 microalga Dunaliella maritima while Na translocating ATPase in the marine microalga Tetraselmis viridis operates as a Na H exchanger .