Triton X-100 as a specific inhibitor of the mammalian NADH-ubiquinone oxidoreductase (Complex I).
Triton X-100 inhibits the NADH oxidase and rotenone-sensitive NADH-Q1 reductase activities of bovine heart submitochondrial particles (SMP) with an apparent Ki of 1x10-5 M (pH 8.0, 25 degrees C). The NADH-hexammineruthenium reductase, succinate oxidase, and the respiratory control ratio with succinate as the substrate in tightly coupled SMP are not affected at the inhibitor concentrations below 0.15 mM. The succinate-supported aerobic reverse electron transfer is less sensitive to the inhibitor (Ki=5x10-5 M) than NADH oxidase. Similar to rotenone, limited concentrations of Triton X-100 increase the steady-state level of NAD+ reduction when the nucleotide is added to tightly coupled SMP oxidizing succinate aerobically. Also similar to rotenone, Triton X-100 partially protects Complex I against the thermally induced deactivation and partially activates the thermally deactivated enzyme. The rate of the NADH oxidase inhibition by rotenone is drastically decreased in the presence of Triton X-100 which indicates a competition between these two inhibitors for a common specific binding site. In contrast to rotenone, the inhibitory effect of Triton X-100 is instantly reversed upon dilution of the reaction mixture. The NADH-Q1 reductase activity of SMP is inhibited non-competitively by added Q1 whereas a simple competition between Q1 and the inhibitor is seen for isolated Complex I. The results obtained show that Triton X-100 is a specific inhibitor of the ubiquinone reduction by Complex I and are in accord with our previous findings which suggest that different reaction pathways operate in the forward and reverse electron transfer at this segment of the mammalian respiratory chain.
Ushakova
A V
AV
Department of Biochemistry, School of Biology, Moscow State University, 119899 Moscow, Russia.
Grivennikova
V G
VG
Ohnishi
T
T
Vinogradov
A D
AD
D000818:Animals; D002211:Capsaicin / Q000031:analogs & derivatives / Q000494:pharmacology; D002417:Cattle; D042967:Electron Transport Complex I; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D017830:Octoxynol / Q000494:pharmacology*; D012402:Rotenone / Q000494:pharmacology
D004791:Enzyme Inhibitors; D012402:Rotenone; D017830:Octoxynol; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; D002211:Capsaicin; C012906:dihydrocapsaicin