Affinity purification of cytochrome c reductase from potato mitochondria.
Ubiquinol-cytochrome-c oxidoreductase has been isolated from potato (Solanum tuberosum L.) mitochondria by cytochrome-c affinity chromatography and gel-filtration chromatography. The procedure, which up to now only proved applicable to Neurospora, yields a highly pure and active protein complex in monodisperse state. The molecular mass of the purified complex is about 650 kDa, indicating that potato cytochrome c reductase occurs as a dimer. Upon reconstitution into phospholipid membranes, the dimeric enzyme catalyzes electron transfer from a synthetic ubiquinol to equine cytochrome c with a turnover number of 50 s-1. The activity is inhibited by antimycin A and myxothiazol. A myxothiazol-insensitive and antimycin-sensitive transhydrogenation reaction, with a turnover number of 16 s-1, can be demonstrated as well. The protein complex consists of ten subunits, most of which have molecular masses similar to those of the nine-subunit fungal enzyme. Individual subunits were identified immunologically and spectral properties of b and c cytochromes were monitored. Interestingly, an additional 'core' polypeptide which is not present in other cytochrome bc1 complexes forms part of the enzyme from potato. Antibodies raised against individual polypeptides reveal that the core proteins are clearly immuno-distinguishable. The additional subunit may perform a specific function and contribute to the high molecular mass which exceeds those reported for other cytochrome-c-reductase dimers.
Braun
H P
HP
Institut für Genbiologische Forschung Berlin GmbH, Federal Republic of Germany.
Schmitz
U K
UK
D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D008081:Liposomes; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D010713:Phosphatidylcholines; D010940:Plant Proteins / Q000302:isolation & purification; D011198:Solanum tuberosum / Q000201:enzymology*
D008081:Liposomes; D010713:Phosphatidylcholines; D010940:Plant Proteins; D009245:NADH Dehydrogenase