Isolation of the alanine carrier from the membranes of a thermophilic bacterium and its reconstitution into vesicles capable of transport.
A carrier protein mediating alanine transport was purified from the membranes of the thermophilic bacterium PS3, by ion exchange chromatography in the presence of both Triton X-100 and urea. The alanine carrier was recovered in the nonadsorbed fraction from either DEAE- or CM-cellulose columns, suggesting that its isoelectric point was in the neutral pH region. The final preparation contained virtually no electron transfer components, ATPase, or NADH dehydrogenase. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the final preparation consisted of two major protein components with molecular weights of 36,000 and 9,400. Active transport of alanine after incorporation of the alanine carrier into reconstituted proteoliposomes was driven not only by an artificial membrane potential generated by potassium ion diffusion via valinomycin but also by mitochondrial cytochrome oxidase incorporated into the same liposomes and supplemented with both cytochrome c and ascorbic acid. The membrane-integrated portion (TFo) of the ATPase complex uncoupled alanine transport by conducting protons across the membrane.
D000409:Alanine / Q000378:metabolism*; D001205:Ascorbic Acid / Q000378:metabolism; D001419:Bacteria / Q000032:analysis* / Q000378:metabolism; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000302:isolation & purification*; D003574:Cytochrome c Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism*; D011092:Polyethylene Glycols; D011510:Proteolipids; D014508:Urea
D002352:Carrier Proteins; D003574:Cytochrome c Group; D008081:Liposomes; D011510:Proteolipids; D011092:Polyethylene Glycols; D014508:Urea; D003576:Electron Transport Complex IV; D000409:Alanine; D001205:Ascorbic Acid