Further studies on the binding of DCCD to cytochrome B and subunit VIII of complex III isolated from beef heart mitochondria.
Complex III (the cytochrome b-c1 complex) from beef heart mitochondria was incubated with [14C]DCCD for various periods of time. The polypeptide profile of the complex was compared in both stained gels and their autoradiograms when three different methods were used to terminate the reaction. Precipitation with ammonium sulfate resulted in the formation of a new band with an apparent molecular weight of 39,000 in both incubated samples and the zero time controls. Reisolation of the complex by centrifugation through 10% sucrose or by precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunit peptides of the complex. Subunit III (cytochrome b) and subunit VIII were the only bands labeled after termination of the reaction by centrifugation through sucrose, while both ammonium sulfate and trichloroacetic precipitation resulted in nonspecific labeling of several other subunits of the complex and increased labeling of subunit VIII relative to subunit III. Preincubation of the complex with antimycin prior to treatment with [14C]DCCD resulted in a 50% decrease in the binding of DCCD to both cytochrome b and subunit VIII. Furthermore, treatment of the complex III with DCCD resulted in a change in the red shift observed after antimycin or myxothiazol addition to the dithionite-reduced complex resulting in a broad peak with no sharp maximum. These results provide further confirmation that DCCD binds preferentially to cytochrome b and subunit VIII of complex III from beef heart mitochondria and suggest that cytochrome b may play a role in proton translocation.
D000818:Animals; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D002234:Carbodiimides / Q000378:metabolism*; D002417:Cattle; D003573:Cytochrome b Group / Q000302:isolation & purification / Q000378:metabolism; D003575:Cytochromes c1 / Q000302:isolation & purification / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism* / Q000494:pharmacology; D014450:Electron Transport Complex III; D008689:Methacrylates; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D011808:Quinone Reductases / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013347:Subcellular Fractions / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology
D002234:Carbodiimides; D003573:Cytochrome b Group; D008689:Methacrylates; D009097:Multienzyme Complexes; D013844:Thiazoles; C032456:antimycin; D004024:Dicyclohexylcarbodiimide; D000968:Antimycin A; C030517:myxothiazol; D003575:Cytochromes c1; D011808:Quinone Reductases; D014450:Electron Transport Complex III