Energy-transducing adenosine triphosphatase from Escherichia coli: purification, properties, and inhibition by antibody.
The membrane adenosine triphosphatase (E.C. 3.6.1.3) from Escherichia coli has been solubilized with Triton X-100 and purified to near homogeneity. The purified enzyme has a sedimentation coefficient of 12.9S in a sucrose gradient, corresponding to a molecular weight of about 360,000. On electrophoresis in gels containing sodium dodecyl sulfate, it dissociates into subunits with apparent molecular weights of 60,000, 56,000, 35,000, and 13,000. The purified enzyme loses activity and breaks down into subunits when stored in the cold. Guanosine 5'-triphosphate and inosine 5'-triphosphate are alternative substrates. Ca(2+) and, to a small extent, Co(2+) or Ni(2+) will substitute for Mg(2+) in the reaction. The K(m) for Mg-adenosine triphosphate of the membrane-bound enzyme is 0.23 mM, and for the pure enzyme it is 0.29 mM. Azide is a noncompetitive inhibitor of both the membrane-bound enzyme and the pure enzyme. P(i) is a noncompetitive inhibitor of the solubilized enzyme. An antibody to the purified enzyme was obtained from rabbits. The antibody inhibits the solubilized enzyme and virtually all of the adenosine triphosphate hydrolysis by membranes from cells grown aerobically or anaerobically. The antibody also inhibits the adenosine triphosphate-stimulated pyridine nucleotide transhydrogenase (E.C. 1.6.1.1) of the E. coli membrane.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D001386:Azides / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D003035:Cobalt / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000276:immunology; D007106:Immune Sera / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009532:Nickel / Q000494:pharmacology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D010759:Phosphorus Isotopes; D011817:Rabbits / Q000276:immunology; D013053:Spectrophotometry; D013501:Surface-Active Agents
D000906:Antibodies; D001386:Azides; D007106:Immune Sera; D010710:Phosphates; D010759:Phosphorus Isotopes; D013501:Surface-Active Agents; D003035:Cobalt; D009532:Nickel; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium