Specific labelling of a constituent polypeptide of bovine heart mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone reductase by the inhibitor diphenyleneiodonium

Ragan, C I; Bloxham, David P.

doi:10.1042/bj1630605

Cited by 112 publications

(55 citation statements)

References 26 publications

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“…1 of Complex I. This behaviour is reminiscent of that of the covalent modifier diphenyleneiodonium (Ragan & Bloxham, 1977), which shows a similar relationship between inhibition and the extent of incorporation of the inhibitor. Titration of mixtures of Complex I and Complex III with antimycin produced similar results to those of Fig.…”

Section: Determination Ofactivation Energiesmentioning

confidence: 60%

The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Evidence for stoicheiometric association

Ragan¹,

Heron²

1978

Biochemical Journal

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1. The NADH-ubiquinone oxidoreductase complex (Complex I) and the ubiquinol-cytochrome c oxidoreductase complex (Complex III) combine in a 1:1 molar ratio to give NADH-cytochrome c oxidoreductase (Complex I-Complex III). 2. Experiments on the inhibition of the NADH-cytochrome c oxidoreductase activity of mixtures of Complexes I and III by rotenone and antimycin indicate that electron transfer between a unit of Complex I-Complex III and extra molecules of Complexes I or III does not contribute to the overall rate of cytochrome c reduction. 3. The reduction by NADH of the cytochrome b of mixtures of Complexes I and III is biphasic. The extents of the fast and slow phases of reduction are determined by the proportion of the total Complex III specifically associated with Complex I. 4. Activation-energy measurements suggest that the structural features of the Complex I-Complex III unit promote oxidoreduction of endogenous ubiquinone-10.

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Section: Determination Ofactivation Energiesmentioning

confidence: 60%

The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Evidence for stoicheiometric association

Ragan¹,

Heron²

1978

Biochemical Journal

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“…Since 0.18/.tM DPI gave full inhibition of NADPH oxidation with 0.44 mg protein (Fig. 3G) the binding of less than l nmol DPI/mg mitochondrial protein, and much less than the 20-30 nmol" (mg protein) -1 required to inhibit Complex I from bovine heart mitochondria [20].…”

Section: Resultsmentioning

confidence: 92%

Direct evidence for the presence of two external NAD(P)H dehydrogenases coupled to the electron transport chain in plant mitochondria

1995

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Exogenous NADPH oxidation by purified mitochondria from both potato tuber and Arum maculatum spadix was completely and irreversibly inhibited by sub-micromolar diphen~leneiodonium (DPI), while exogenous NADH oxidation was inhibited to only a small degree. Addition of DPI caused the collapse of the membrane potential generated by NADPH oxidation, while the potential generated by NADH was unaffected. We conclude that there are two distinct enzymes on the outer surface of the inner membrane of plant mitochondria, one specific for NADH, the other relatively specific for NADPH, with both enzymes linked to the electron transport chain.Key words: Diphenyleneiodonium; Electron transport chain; Mitochondria, plant; NAD(P)H dehydrogenase |. IntroductionUnlike mammalian mitochondria, the electron transport hain of plant mitochondria contains at least two NAD(P)H ,tehydrogenases in addition to Complex I [1]. One of these ~otenone-insensitive dehydrogenases is located on the outer , urface of the inner mitochondrial membrane, the other on the inner, matrix surface, and in the following we will refer to them ;s NDex and NDin, respectively.Due to the presence of NDex, plant mitochondria oxidize ~xogenous NADH and NADPH. The resulting electrons enter he electron transport chain at ubiquinone bypassing Complex i, the site of rotenone inhibition [1][2][3][4][5]. Compared to NADH ~xidation, NADPH oxidation has a lower pH optimum [6][7][8], s more sensitive to thiol reagents [7,9] and appears to require nore Ca 2÷ for activity [9][10][11][12]. NADH oxidation has been re~orted to be induced in red beetroots without a concomitant nduction of NADPH oxidation [13]. Likewise, mitochondria rom suspension-cultured cells of sugar beet oxidize NADH, ~ut exhibit very low rates of NADPH oxidation [14]. On the )asis of this circumstantial evidence it has been concluded that t is most likely that two separate enzymes exist, one for each ,:oenzyme [2,4,14]. However, no direct evidence has ever been r~resented to support this conclusion.Diphenyleneiodonium ( Materials and methodsMitochondria were prepared from potato (Solanum tuberosum L.) tubers according to [16]. Crude mitochondria were isolated from Arum maculatum spadices according to [17] and purified according to [18].Oxygen consumption was measured in a medium containing 0.3 M sucrose, 5 mM MOPS (pH 7.2), 5 mM KH:PO4, 2.5 mM MgCI2, 1 mM CaC12 and 0.4 ¢tM FCCP using an oxygen electrode (Rank Bros.). The membrane of the oxygen electrode was washed with 70% (v/v) ethanol between assays to avoid carry-over of DPI.Membrane potential measurements using 16 ]zM safranine as the probe were made according to [19]. The medium used was the same as that above except that FCCP was omitted, CaCI2 was 0.1 mM and 20 ,ug.mg 1 BSA was included.NAD(P)H oxidation with oxygen as the electron acceptor was assayed spectrophotometrically at 340 nm in the same medium used for the oxygen electrode measurements. Activities were calculated using an extinction coefficient for NAD(P)H of 6.2 mM -1 .cm -~, All ...

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“…This may be due to the high proliferative rate of N11 cells as cell division needs huge amounts of NADPH and ribose-5-phosphate for nucleotide synthesis. The inhibition of the tricarboxylic acid cycle could be accounted for by the inhibitory effect of DPI on the cell respiration (2,3). The inhibition of the PPP by DPI or DIP has been already observed in several cell types, such as hepatic endothelial cells (31) and Kupffer cells (32) in rats treated with endotoxin in vivo and rat peritoneal macrophages stimulated with phorbol myristate acetate (33).…”

Section: Resultsmentioning

confidence: 99%

Diphenyleneiodonium Inhibits the Cell Redox Metabolism and Induces Oxidative Stress

Riganti

Gazzano

Polimeni

et al. 2004

Journal of Biological Chemistry

178

133

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Diphenyleneiodonium (DPI) and the structurally related compound diphenyliodonium (DIP) are widely used as inhibitors of flavoenzymes, particularly NADPH oxidase. Here we report further evidence that DPI and DIP are not specific flavin binders. A 3-h incubation of N11 glial cells with DPI significantly inhibited in a dosedependent way both the pentose phosphate pathway and the tricarboxylic acid cycle. In parallel, we observed a dose-dependent increase of reactive oxygen species generation and lipoperoxidation and increased leakage of lactate dehydrogenase activity in the extracellular medium. The glutathione/glutathione disulfide ratio decreased, whereas the efflux of glutathione out of the cells increased. This suggests that DPI causes an augmented oxidative stress and exerts a cytotoxic effect in N11 cells. Indeed, the cells were protected from these events when loaded with glutathione. Similar results were observed using DIP instead of DPI and also in other cell types. We suggest that the DPI-elicited inhibition of the pentose phosphate pathway and tricarboxylic acid cycle may be mediated by the blockade of several NAD(P)-dependent enzymes, such as glucose 6-phosphate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase. In light of these results, we think that some effects of DPI or DIP in in vitro and in vivo experimental models should be interpreted with caution. Diphenyleneiodonium (DPI) 1 and the structurally related compound diphenyliodonium (DIP) are widely used as uncompetitive inhibitors of flavoenzymes. Firstly identified as a hypoglycemic agent able to block gluconeogenesis and respiration in rat liver (1), DPI has been subsequently shown to inhibit the activity of NADH:ubiquinone oxidoreductase (2, 3), NADPH oxidase (4 -6), nitric-oxide synthase (7), xanthine oxidase (6), and NADPH cytochrome P450 oxidoreductase (8). DPI and other iodonium derivatives have been shown to react via a radical mechanism, whereby an electron is abstracted from FAD or FMN to form a radical, which then adds back to the flavin to form covalent, phenylated adducts (9). Also, heme groups, such as the heme b of NADPH oxidase, have been found to react with DPI and DIP (10).In most experimental works of the last years, DPI or DIP have been used as inhibitors of NADPH oxidase. NADPH oxidases are a group of plasma membrane-associated enzymes found in a variety of cells of mesodermal origin. The most thoroughly studied is the leukocyte isoform, which catalyzes the production of superoxide (O 2 . ) by the one-electron reduction of oxygen, using NADPH as the reducing agent (11). The O 2 .generated by NADPH oxidase serves as the starting material for the production of a vast assortment of reactive oxidants used by phagocytes to kill invading microorganisms or tumor cells (11). A low activity NADPH oxidase is present in a variety of nonphagocytic cells, wherein this enzyme is a source of second messengers. It has been postulated, for instance, that the O 2 . generated by the aorta functions as a blood ...

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Specific labelling of a constituent polypeptide of bovine heart mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone reductase by the inhibitor diphenyleneiodonium

Cited by 112 publications

References 26 publications

The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Evidence for stoicheiometric association

The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Evidence for stoicheiometric association

Direct evidence for the presence of two external NAD(P)H dehydrogenases coupled to the electron transport chain in plant mitochondria

Diphenyleneiodonium Inhibits the Cell Redox Metabolism and Induces Oxidative Stress

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