Isolation of mitochondrial succinate: ubiquinone reductase, cytochrome c reductase and cytochrome c oxidase from Neurospora crassa using nonionic detergent.
The electron transfer complexes, succinate: ubiquinone reductase, ubiquinone: cytochrome c reductase, and cytochrome c: O2 oxidase were isolated from the mitochondrial membranes of Neurospora crassa by the following steps. Modification of the contents of the complexes in mitochondria by growing cells on chloramphenicol; solubilisation of the complexes by Triton X-100; affinity chromatography on immobilized cytochrome c and ion exchange and gel chromatography. Ubiquinone reductase was obtained in a monomeric form (Mr approximately 130 000) consisting of a flavin subunit (Mr 72 000) an iron-sulfur subunit (Mr 28 000) and a cytochrome b subunit (Mr probably 14 000). Cytochrome c reductase was obtained in a dimeric form (Mr approximately 550 000), the monomeric unit comprising the cytochromes b (Mr each 30 000), a cytochrome c1 (Mr 31 000), the iron-sulfur subunit (Mr 25 000), and six subunits without known prosthetic groups (Mr 9000, 11 000, 14 000, 45 000, 45 000, and 52 000). Cytochrome c oxidase was also isolated in a dimeric form (Mr approximately 320 000) comprising two copies each of seven subunits (Mr 9000, 12 000, 14 000, 18 000, 21 000, 29 000, and 40 000). The complexes were essentially free of phospholipid. Each bound one micelle of Triton X-100 (Mr approximately 90 000). After isolation, the bound Triton X-100 could be replaced by other nonionic detergents such as: alkylphenyl polyoxyethylene ethers, alkyl polyoxyethylene ethers and acyl polyoxyethylene sorbitan esters.
D002701:Chloramphenicol / Q000494:pharmacology; D003579:Cytochrome Reductases / Q000302:isolation & purification*; D003574:Cytochrome c Group; D003576:Electron Transport Complex IV / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D009245:NADH Dehydrogenase / Q000302:isolation & purification*; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000187:drug effects / Q000201:enzymology*; D011092:Polyethylene Glycols; D011808:Quinone Reductases / Q000302:isolation & purification*; D012995:Solubility; D013053:Spectrophotometry; D014451:Ubiquinone
D003574:Cytochrome c Group; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D014451:Ubiquinone; D011092:Polyethylene Glycols; D002701:Chloramphenicol; D009247:NADH, NADPH Oxidoreductases; D003579:Cytochrome Reductases; D011808:Quinone Reductases; D009245:NADH Dehydrogenase; D003576:Electron Transport Complex IV