title: Dicyclohexylcarbodiimide binds to cytochrome b and subunit VIII in soluble complex III from beef heart mitochondria.
abstract: Incubation of soluble complex III isolated from either yeast or beef heart mitochondria with 25-100 nmol of [14C]dicyclohexylcarbodiimide (DCCD)/nmol of cytochrome b followed by centrifugation through 10% sucrose or precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunits of either complex. The [14C]DCCD was bound to cytochrome b and phospholipids in the yeast complex and with similar kinetics to both cytochrome b and subunit VIII (Mr = 4000-8000) plus phospholipids of the beef complex. Subunit VIII of the beef complex was partially extracted with chloroform:methanol; however, no subunit of this mobility was present in the yeast complex. Incubation of the beef complex in phosphate buffer for short times resulted in a doubling of the [14C]DCCD bound to cytochrome b relative to that to subunit VIII. Preincubation of both complexes with venturicidin prior to treatment with DCCD resulted in a 50% decrease in the binding of [14C]DCCD to cytochrome b. Reisolation of the beef complex III by precipitation with (NH4)2SO4 after incubation with [14C]DCCD resulted in the formation of a new band with an apparent molecular weight of 39,000 even in the zero time control. The [14C]DCCD was bound to subunit VIII and the core proteins but not to cytochrome b at all times, suggesting that precipitation with (NH)2SO4 in the presence of DCCD causes cross-linking of the subunits of complex III.
mesh_terms: D000645:Ammonium Sulfate; D000818:Animals; D001345:Autoradiography; D002234:Carbodiimides / Q000378:metabolism*; D002417:Cattle; D003573:Cytochrome b Group / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D014450:Electron Transport Complex III; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011808:Quinone Reductases / Q000378:metabolism*; D014698:Venturicidins / Q000494:pharmacology; D015003:Yeasts / Q000201:enzymology
chemical_list: D002234:Carbodiimides; D003573:Cytochrome b Group; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D014698:Venturicidins; D004024:Dicyclohexylcarbodiimide; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D014450:Electron Transport Complex III; D000645:Ammonium Sulfate