Solubilization and reconstitution of theRickettsia prowazekii ATP/ADP translocase
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The ATP/ADP translocase protein ofRickettsia prowazekii, an obligate intracellular parasite that had been grown in the chick yolk sac, was solubilized and reconstituted into liposomes composed ofEscherichia coli phospholipid by an octylglucoside dilution procedure. Proteoliposomes prepared from membranes of Renografin-purifiedR. prowazekii translocated ATP by an obligate exchange mechanism. Influx of extravesicular ATP required intravesicular transportable nucleotide and efflux of intravesicular ATP required transportable extravesicular nucleotide in the medium. The transport activity was insensitive to carboxyatractyloside and bongkrekic acid, inhibitors of mitochondrial ADP/ATP translocation. Proteoliposomes prepared from membranes of standard (non-Renografin-purified)R. prowazekii exhibited both an inhibitor-sensitive mitochondrial translocase activity and an inhibitor-resistant rickettsial translocase activity. Proteoliposomes prepared from uninoculated yolk sac membranes exhibited only the inhibitor-sensitive mitochondrial translocase activity. The substrate specificity of each reconstituted translocase was determined and shown to correspond with that reported for intact mitochondria or rickettsiae. Following influx of ATP the steady-state value for intravesicular labeled ATP was dependent on the concentration of intravesicular nucleotide available for exchange.
Key Wordsproteoliposomes ATP/ADP translocase Rickettsia prowazekii reconstitution octylglucoside mitochondria
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