Abstract
At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.
Highlights
At the concentrations usually employed as a Ca2+ compounds have been shown tobe reduced both in vivo and indicator, arsenazo 111underwent a one-electron re- in vitro by enzymes localized mainly in the microsomal fracduction by rat liver mitochondria to produce an azo tion of liver [2,3,4,5,6,7,8],with theenzyme responsible for azo reducanion radical as demonstrated by electron-spin reso- tion appearing to depend upon the substrate
Our studies showed that intact ratliver mitochondria were able to reduce arsenazo 111 enzymatically to an anion free radical metabolite whenever reduced pyridine nucleotides were present in the incubation mixtures
NADH-dependent arsenazo 111reduction seems to be catalyzed by an outer membrane azoreductase because: (a) the outer mitochondrial membrane, where the azoreductase is apparently located, was freely accessible to exogenous NADH (Fig. 4); ( b ) the azoreductase activity was not supported by endogenous NADH, as shown in Fig. 4, unless the mitochondrial membranes were solubilized; (c) the activity was not decreased by rotenone, antimycin A, or KCN (Table I); and ( d ) the activity was higher in crude outer membrane preparations than in mitoplasts or whole mitochondria (Table 11)
Summary
In compounds [7, 8], and xanthine oxidase has been postulated addition, NAD(P)H azoreductase activity was higher [9] tocatalyze azo reduction. In addition to their wide use in the pharmaceutical, food, thanin mitoplasts andintact mitochondria. The suspension was centrifuged a t 8,000 X g for 10 min and the pellet was resuspended in a buffer consisting of 0.25 M sucrose, 15 mM MgC12,and 40 mM Tris-HC1, pH. For the experiments with submitochondrial particles (ESR and oxygen consumption measurements), the reaction mixture contained protein (1 mg/ml), 100 p M arsenazo 111, and 5 mM succinate in the reaction buffer described above. The instrument was set in the split-beam mode, and the complete incubation, with the exception of NAD(P)H, was put into both the sample and reference sides
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