A study of the dicyclohexylcarbodiimide-binding component of the mitochondrial ATPase complex from beef heart.
1. The binding of [14C]-dicyclohexylcarbodiimide to membrane proteins of beef heart mitochondria has been investigated using dodecylsulphate/polyacrylamide gel electrophoresis. Upon incubation of submitochondrial particles with low concentrations of dicyclohexylcarbodiimide (5 nmol/mg protein) radioactivity was incorporated into three components with apparent molecular weights of 30000, 18000 and less than 6500. Only the two smaller components were found to be extracted into chloroform/methanol. The same two components were labelled when the isolated ATPase complex or a reconstituted F0F1 system was incubated with low concentrations of dicyclohexylcarbodiimide. High concentrations of dicyclohexylcarbodiimide (20-100 nmol/mg protein) resulted in binding to several mitochondrial proteins. 2. The maximal amount of dicyclohexylcarbodiimide which can bind to submitochondrial particles, the isolated ATPase complex, and the reconstituted F0F1 system was found to exceed the amount required for maximal inhibition of the ATPase activity by several-fold. The distribution of the bound [14C]dicyclohexylcarbodiimide between the different dicyclohexylcarbodiimide-binding components was investigated as a function of dicyclohexylcarbodiimide concentration. The smallest and largest components revealed a high affinity for dicyclohexylcarbodiimide-binding which paralleled the inhibition of ATPase activity. The intermediate component had a markedly lower affinity for dicyclohexylcarbodiimide-binding. 3. The larger dicyclohexylcarbodiimide-binding component of the isolated ATPase complex can be converted into the smaller component by treatment of the ATPase complex with performic acid. Partial conversion can also be achieved by extraction of the band from the dodecylsulphate-polyacrylamide gel after electrophoresis, followed by re-electrophoresis. The observations suggest that the larger component may be an oligomer of the smaller one. 4. Using concentrations of oligomycin and dicyclohexylcarbodiimide which were equal to or greater than those required for maximal inhibition of the ATPase activity, oligomycin was found to diminish the binding of [14C]dicyclohexylcarbodiimide to both dicyclohexylcarbodiimide-binding components of the isolated ATPase complex.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000818:Animals; D001665:Binding Sites; D002234:Carbodiimides / Q000378:metabolism*; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000302:isolation & purification; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight; D009840:Oligomycins / Q000494:pharmacology; D011485:Protein Binding; D012967:Sodium Dodecyl Sulfate
D002234:Carbodiimides; D008565:Membrane Proteins; D009840:Oligomycins; D012967:Sodium Dodecyl Sulfate; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases