Binding of detergents and inhibitors to bovine complex I - a novel purification procedure for bovine complex I retaining full inhibitor sensitivity.
Mitochondrial complex I exhibits some peculiar and poorly understood features regarding the effects of detergents on activity and sensitivity to hydrophobic inhibitors that are not seen with other membrane complexes using ubiquinone as a substrate. Therefore, we investigated the interaction of complex I from bovine heart mitochondria with different types of detergents by monitoring activity, degree of inhibition and inhibitor binding in the presence of increasing concentrations of detergent. It is shown that apart from their nature as solubilizing and delipidating agents the polyoxyethylene-ether detergents Triton X-100, Brij-35 and Thesit act as specific inhibitors of complex I and compete with classical complex I inhibitors for a common binding domain. These findings were used to develop a novel large-scale chromatographic procedure for isolation of inhibitor-sensitive NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxylapatite, ion-exchange and gel-exclusion chromatography. By switching detergents from Triton X-100 to dodecylmaltoside after hydroxylapatite chromatography the procedure yields highly pure, monodisperse and fully inhibitor-sensitive enzyme.
Universitätsklinikum Frankfurt, Institut für Biochemie I, Zentrum der Biologischen Chemie, Frankfurt am Main, Germany.
J G
JG
Okun
V
V
Zickermann
K
K
Zwicker
H
H
Schägger
U
U
Brandt
D000588:Amines / Q000737:chemistry; D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D013696:Temperature
D000588:Amines; D003902:Detergents; D004791:Enzyme Inhibitors; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I