Reconstitution of oxidative phosphorylation and the adenosine triphosphate-dependent transhydrogenase activity by a combination of membrane fractions from unCA- and uncB- mutant strains of Escherichia coli K12.
1. Membrane preparations from both uncA(-) and uncB(-) mutant strains of Escherichia coli K12, in which electron transport is uncoupled from phosphorylation, were fractionated by washing with a low-ionic-strength buffer. The fractionation gave a ;5mm-Tris wash' and a ;membrane residue' from each strain. This technique, applied to membranes from normal cells, separates the Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity from the membrane-bound electron-transport chain and the non-energy-linked transhydrogenase activity. 2. Reconstitution of both oxidative phosphorylation and the ATP-dependent transhydrogenase activity was obtained by a combination of the ;membrane residue' from strain AN249 (uncA(-)) with the ;5mm-Tris wash' from strain AN283 (uncB(-)). 3. Valinomycin plus NH(4) (+) inhibited oxidative phosphorylation both in membranes from a normal strain of E. coli and in the reconstituted membrane system derived from the mutant strains. 4. The electron-transport-dependent transhydrogenase activity was located in the membrane residue and was de-repressed in both the mutant strains. 5. The spatial and functional relationships between the proteins specified by the uncA and uncB genes and the transhydrogenase protein are discussed.
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D000641:Ammonia / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D004579:Electron Transport; D004926:Escherichia coli / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D010085:Oxidative Phosphorylation; D010088:Oxidoreductases / Q000032:analysis*; D014161:Transduction, Genetic; D014461:Ultracentrifugation; D014634:Valinomycin / Q000494:pharmacology
D014634:Valinomycin; D000641:Ammonia; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium