Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase.
1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 2. The Mg(2+),Ca(2+)-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA(-)) could not be re-activated by the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA(-)).
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate; D002118:Calcium; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D002850:Chromatography, Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology*; D005809:Genes, Regulator; D007700:Kinetics; D008274:Magnesium; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism*; D011134:Polysaccharides; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D014461:Ultracentrifugation; D014462:Ultrafiltration
D011134:Polysaccharides; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium