Catalytic activity of NADH-ubiquinone oxidoreductase (complex I) in intact mitochondria. evidence for the slow active/inactive transition.
The mammalian purified dispersed NADH-ubiquinone oxidoreductase (Complex I) and the enzyme in inside-out submitochondrial particles are known to be the slowly equilibrating mixture of the active and de-activated forms (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169-185). We report here the phenomenon of slow active/de-active transition in intact mitochondria where the enzyme is located within its natural environment being exposed to numerous mitochondrial matrix proteins. A simple procedure for permeabilization of intact mitochondria by channel-forming antibiotic alamethicin was worked out for the "in situ" assay of Complex I activity. Alamethicin-treated mitochondria catalyzed the rotenone-sensitive NADH-quinone reductase reaction with exogenousely added NADH and quinone-acceptor at the rates expected if the enzyme active sites would be freely accessible for the substrates. The matrix proteins were retained in alamethicin-treated mitochondria as judged by their high rotenone-sensitive malate-cytochrome c reductase activity in the presence of added NAD(+). The sensitivity of Complex I to N-ethylmaleimide and to the presence of Mg(2+) was used as the diagnostic tools to detect the presence of the de-activated enzyme. The NADH-quinone reductase activity of alamethicin-treated mitochondria was sensitive to neither N-ethylmaleimide nor Mg(2+). After exposure to elevated temperature (37 degrees C, the conditions known to induce de-activation of Complex I) the enzyme activity became sensitive to the sulfhydryl reagent and/or Mg(2+). The sensitivity to both inhibitors disappeared after brief exposure of the thermally de-activated mitochondria with malate/glutamate, NAD(+), and cytochrome c (the conditions known for the turnover-induced reactivation of the enzyme). We conclude that the slow active/de-active Complex I transition is a characteristic feature of the enzyme in intact mitochondria and discuss its possible physiological significance.
Grivennikova
V G
VG
Department of Biochemistry, School of Biology, Moscow State University, Moscow 119899, Russian Federation.
Kapustin
A N
AN
Vinogradov
A D
AD
D000408:Alamethicin / Q000494:pharmacology; D000818:Animals; D002384:Catalysis; D002417:Cattle; D042967:Electron Transport Complex I; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D051381:Rats
D000408:Alamethicin; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I