Purification and properties of a dicyclohexylcarbodiimide-sensitive adenosine triphosphatase from a thermophilic bacterium.
1. A stable ATPase complex with sensitivity to dicyclohexylcarbodiimide (TFo-F1) was purified from the membranes of the thermophilic aerobic bacterium PS3, by ion exchange chromatography in the presence of Triton X-100. 2. The ATPase of TFo-F1 was maximal at 70 degrees at pH 8.6 and was stable after monomerization in 4 M urea and 0.5% Triton X-100 at 25 degrees. The activity was dependent on Mg2+, Co2+, or Mn2+, and it became insensitive to dicyclohexylcarbodiimide when Ca2+ or Cd2+ was added instead. 3. TFo-F1 required P-lipids of this bacterium contained branched fatty acyl groups but no unsaturated groups and were stable against oxidation and heat. 4. Studies by electron microscopy, gel electrophoresis, and use of anti-ATPase antibody and [3H]acetyl-ATPase indicated that the TFo-F1 complex was composed of an ATPase moiety (TF1, five different subunits) and a hydrophobic moiety (TFo, three different subunits. TFo conferred TF1 with sensitivity to dicyclohexylcarbodiimide. 5. Vesicles catalyzing 32Pi-ATP exchange and ATP-driven enhancement of fluorescence of anilinonaphthalene sulfonate were reconstituted by dialyzing pure TFo-F1 and P-lipids together, and were active even at 50-75 degrees. The vesicles reconstituted from TFo-F1 and bacterial P-lipids were more stable than those reconstituted from TFo-F1 and soybean P-lipids.
D000251:Adenosine Triphosphatases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates; D001419:Bacteria / Q000201:enzymology*; D002234:Carbodiimides / Q000494:pharmacology*; D002413:Cations, Divalent; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004355:Drug Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D010743:Phospholipids / Q000494:pharmacology; D011092:Polyethylene Glycols; D014475:Uncoupling Agents / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D002234:Carbodiimides; D002413:Cations, Divalent; D046911:Macromolecular Substances; D010743:Phospholipids; D014475:Uncoupling Agents; D011092:Polyethylene Glycols; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases