Time and concentration dependence of the dicyclohexylcarbodiimide inhibition of proton movements in the cytochrome bc1 complex from yeast mitochondria reconstituted into proteoliposomes.
The cytochrome bc1 complex was isolated from yeast mitochondria solubilized with the detergent dodecyl maltoside and reconstituted into proteoliposomes to measure electrogenic proton pumping. Optimal respiratory control ratios of 4.0, obtained after addition of the uncoupler CCCP, and H+/e- ratios of 1.6 were obtained when the proteoliposomes were prepared with egg yolk phosphatidylcholine supplemented with cardiolipin. Moreover, it was critical to remove excess dodecyl maltoside in the final concentrated preparation prior to reconstitution to prevent loss of enzymatic activity. The rate of electrogenic proton pumping, the respiratory control ratios, and the H+/e- ratios were decreased by incubation of the cytochrome bc1 complex with dicyclohexylcarbodiimide (DCCD) in a time and concentration dependent manner. Maximum inhibitions were observed when 50 nmol DCCD per nmol of cytochrome b were incubated for 30 min at 12 degrees C with the intact cytochrome bc1 complex. Under these same conditions maximum labeling of cytochrome b with [14C] DCCD was reported in a previous study [Beattie et al. (1984). J. Biol. Chem. 259, 10562-10532] consistent with a role for cytochrome b in electrogenic proton movements.
Beattie
D S
DS
Department of Biochemistry, West Virginia University School of Medicine, Morgantown 26506.
Marcelo-Baciu
R M
RM
D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D011510:Proteolipids / Q000378:metabolism*; D011522:Protons; D015003:Yeasts / Q000201:enzymology
D008081:Liposomes; D011510:Proteolipids; D011522:Protons; C020485:proteoliposomes; D004024:Dicyclohexylcarbodiimide; D014450:Electron Transport Complex III