Purification and preliminary characterization of mitochondrial complex I (NADH: ubiquinone reductase) from broad bean (Vicia faba L.).
NADH:ubiquinone reductase (EC 1.6.19.3), or complex I, was isolated from broad bean (Vicia faba L.) mitochondria. Osmotic shock and sequential treatment with 0.2% (v/v) Triton X-100 and 0.5% (w/v) [3-cholamidopropyl)dimethylammonio]-1-propanesulfate (CHAPS) removed all other NADH dehydrogenase activities. Complex I was solubilized in the presence of 4% Triton X-100 and then purified by sucrose-gradient centrifugation in the presence of the same detergent. The second purification step was hydroxylapatite chromatography. Substitution of CHAPS for Triton X-100 helped remove contaminants such as ATPase. The high molecular mass complex is composed of at least 26 subunits with molecular masses ranging from 6000 to 75,000 kD. The purified complex I reduced ferricyanide and ubiquinone analogs but not cytochrome c. NADPH could not substitute for NADH as an electron donor. The KM for NADH was 20 microM at the optimum pH of 8.0. The NH2-terminal sequence of several subunits was determined, revealing the ambiguous nature of the 42-kD subunit.
Leterme
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Unité de Biochimie Physiologique, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.
Boutry
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D000595:Amino Acid Sequence; D000818:Animals; D002793:Cholic Acids; D003902:Detergents; D042967:Electron Transport Complex I; D007887:Fabaceae / Q000201:enzymology* / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010946:Plants, Medicinal; D011487:Protein Conformation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate