ATP-dependent proton uptake by proteoliposomes reconstituted with purified Na+,K+-ATPase.
Proton transport catalyzed by the sodium pump was demonstrated using proteoliposomes reconstituted with purified pig kidney Na+,K+-ATPase. Intravesicular pH was monitored with fluorescence from fluorescein isothiocyanate dextran introduced into the vesicles. An ATP-induced ouabain-sensitive acidification of the intravesicular medium was observed, when the vesicles were incubated with ATP and without Na+. The ATP-induced acidification was blocked by either extravesicular Na+ or pretreatment of the enzyme with ouabain before reconstitution. Protonophores, X-537A or carbonyl cyanide m-chlorophenylhydrazone, abolished the intravesicular acidification. The acidification was not inhibited by 3 mM tetra-n-butylammonium. The initial rate of the H+ uptake was increased with a decrease in pH of the extravesicular medium, and the maximum rate was obtained at pH 5.5-5.6. It is concluded that H+ can be transported in place of Na+ by the sodium pump.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D003911:Dextrans; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D008564:Membrane Potentials; D010042:Ouabain / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism*; D011522:Protons; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D003911:Dextrans; D005452:Fluoresceins; D011510:Proteolipids; D011522:Protons; C015219:fluorescein isothiocyanate dextran; C020485:proteoliposomes; D010042:Ouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate