Biochem. J. (1976) 154, 307-310

Printed in Great Britain

Ineractions of Some Acceptors with Superoxide Anion Radicals Formned by the NADPPH-Specific Flavoprotein in Rat Liver Microsomal Fractions By VLADIMIR MISHIN, ANDREY POKROVSKY and VYACHESLAV V. LYAKHOVICH Department of Biochemistry, Institute of Clinical and Experimental Medicine, Academy of Medical Sciences of the U.S.S.R., Siberian Branch, Novosibirsk 630091, U.S.S.R. (Received 5 August 1975)

Inrat liver microsomal fractions oxidation of adrenaline was effected by superoxide anion radicals (A- ), whereas cytochrome c, 2,6-dichlorophenol- indophenol and ferricyanide accepted electrons from NADPH-specific filavoprotein only directly. Nitro Blue Tetrazolium was reduced both by 02- and by tbe direct acceptance of electrons. Elevation of pH and addition of menadione shift the Nitro Blue Tetrazolium reduction towards the O2-dependent pathway. From the values of the kinetic constants for interaction of adrenaline and Nitro Blue Tetrazolium with NADPH-specific flavoprotein, the rates of generation of 02- in rat liver microsomal fraction were determined. The flavoprotein NADPH-cytochrome c reductase (EC 1,6.2.4) of the NADPHITspecific microsomnal electron-transport chain reduces various acceptors which can be classified into two groups. Some acceptors take up the electrons directly from the flavoprotein and are capable of being reduced in the absence of 02, namely cytochrome c (Masters et at., 1965), ferriiyanide (Lyanagi et al., 1974) and the natural electron acceptor, cytochrom P-450 (Gnosspelius et al., 1969), which catalyses the xenobiotic hydroxylation reactions. The second group includes adrenaline, which, as demonstrated by Aust et a!. (1972), is oxidized by the liver microsomal fractions to yield adrenochrome under the action of superoxide anion radicals (02-'), supplied by NADPH-cytochrome c reductase. In this case, the electrons from the flavoprotein accept the oxygen molecule to form 02-, which subsequently reacts with adrenaline. The importance of studying the generation of 02-

is obvious, since it has been shown that 02-' has a damaging effect on, the cellular membrane (Pederson & Aust, 1972; Fong etal., 1973), and also participates in the hydroxylation reaction in model systems (Prema Kumar et al., 1972). The aim of the present study was to investigate the interaction of NADPH-specific flavoprotein in liver microsomal fraction with oxygen by using for this purpose acceptors that react with 02- formed by NADPH-cytochrome c reductase.

Experimental Male Wistar rats weighing 150-200g were starved for 18h before the experiment and killed by decapitaVol. 154

tion. The liver was perfused in situ with ice-cold isolation medium: 1.15% (w/v) KCI/0.02M-Tris/HCI, pH7A. The microsomal fraction was prepared by differential centrifugation (Tsyrlov et al., 1972). The protein in the microsomal suspension was determined by the biuret method (Gornall et al., 1949). Reduction of 2,6-dichlorophenol-indophenol was measured at 600nm, by using e = 21000 litre mol-h cm-' (Steyn.Parve & Beinert, 1958), and 100puM2,6-dichlorophenol-indophenol. Reduction of ferricyanide


measured at 420nm, by using


litre mol-h cm-' (Schatz, 1967), and 50uM-ferricyanide. Adrenaline oxidation was assayed by the rate of adrenochrome formation at 480nm, by using e = 4020 litre mol-h cm-' (Green et al., 1956), and adrenaline at an initial concentration of 300uM. Reduction of cytochrome c and Nitro Blue Tetrazolium was measured as described previously (Phillips & Langdon, 1962; Roering et al., 1972). NAJPH oxidation was measured by following the decrease in F340, Spectral change$ were mesured with a Hitachi 356 spectrophotpmeter. The assay medium contained 0.15M-potassium phosphate buffer, pH8.0, 1004UM-EDTA, 1004uM-NADPH, 0.4-0.5 mg of micro-

somal protein/ml, the acceptors at the indicated concentrations, at 220C in a cuvette of'volume 3ml. Superoxide dismutase isolated from bovine erythrocytes (McCord & Fridovich, 1969) was used in the experiments. Results We studied electron acceptors of microsomal NADPH-specific flavoprotein to identify the



reactions with 02-. The criterion governing the involvement of 02-- in these reactions was the inhibition by superoxide dismutase, which catalyses O2dismutation (McCord & Fridovich, 1969). It was found that superoxide dismutase inhibited Nitro Blue Tetrazolium reduction in liver microsomal fractions, whereas it did not affect the reduction of other acceptors under study (Table 1). Partial blocking of Nitro Blue Tetrazolium reductase indicates that Nitro Blue Tetrazolium is reduced by liver microsomal fractions in two pathways: (a) the electrons are accepted directly from the flavoprotein (02--independent pathway, superoxide dismutase exercises no effect); (b) electrons accepted from 02-radicals, which are generated by NADPH-cytochrome c reductase (02--dependent pathway, inhibited by superoxide dismutase). Also investigated was the dependence of the inhibition of adrenaline oxidation and Nitro Blue Tetrazolium reduction on superoxide dismutase concentration. A maximum

Nitro Blue Tetrazolium reductase inhibition of 20% was achieved after adding 2,ug of superoxide dismutase/ml. Oxidation of adrenaline to adrenochrome, induced by liver microsomal fractions, was completely blocked by this concentration of superoxide dismutase. It is noteworthy that a 50% inhibition of this reaction occurred at a concentration of 1.2,ug/ml of the enzyme used by us. Superoxide dismutase inactivated by boiling for 3 min had no inhibiting effect on Nitro Blue Tetrazolium reduction and adrenaline oxidation by liver microsomal fractions. When examining the influence of pH on the reduction rate of Nitro Blue Tetrazolium, cytochrome c and adrenaline oxidation it was discovered that an increase in pH increased the rate of 02--dependent reactions of Nitro Blue Tetrazolium and adrenaline, whereas the rate of °2--independent reduction of Nitro Blue Tetrazolium and cytochrome c practically remained unaltered (Table 2). The information presented in Table 3 indicates that

Table 1. Effect of superoxide dismutase on the reduction of various acceptors in rat liver microsomalfractions Experiments were carried out with an assay medium containing 0.15M-potassium phosphate buffer, pH8.0, 1 00gCM-EDTA, 1OO1uM-NADPH, 0.4-0.5mg of microsomal protein/ml, at 22°C, in a cuvette of volume 3ml. The concentrations of acceptors are indicated in the Experimental section. The menadione concentration (column 5) was 20pM. Enzyme activity

(nmol of acceptor reduced/min per mg of protein)

Control +Superoxide dismutase

Menadione reductases Nitro Blue (nmol of NADPH Cytochrome c Ferricyanide Tetrazolium 2,6-Dichlorophenol- oxidized/min per mg reductase reductase indophenol reductase of protein) reductase 72.0 81.0 174 38.5 15.4 83.2 72.5 174 15.3 31.0

(lSg/ml) +Superoxide dismutase






(4Ogg/ml) Table 2. Effect ofpHon rate of interaction between rat liver microsomalfractions and Nitro Blue Tetrazolium, adrenaline an cytochrome c Experimental conditions as described in Table 1. The 'common rate' for Nitro Blue Tetrazolium reductase activity is the reaction rate without the superoxide dismutase addition. The inhibition of adrenaline oxidase by superoxide dismutase (I5,ug/ml) was complete at any pH. Enzyme activity

(nmol of acceptor reduced/min per mg of protein) Nitro Blue Tetrazolium reductase pH 7.5 8.0 8.5


02 -dependent

rate' 31.9 37.0 38.4


02- -dependent rate (as % of 'common rate')

2.2 7.5 9.6

7 20 25

Cytochrome c reductase 68.0 68.4 69.1

Adrenaline oxidase (nmol of adrenochrome formed/min per mg of protein) 8.2 12.4 15.7 1976



Table 3. Effect ofmenadione on adrenaline oxidation and Nitro Blue Tetrazolium reductase Enzyme activities are expressed as in Tables 1 and 2. The menadione concentration was 20juM. Enzyme activity Nitro Blue Tetrazolium reductase I

Control +Menadione

Adrenaline oxidase 12.0 24.6

'Common rate' 37.0 37.0

02j-dependent rate

7.5 15.3

Inhibition by superoxide dismutase (154ug/ml) (Y.) 20 40

Table 4. Kinetic constants for Nitro Blue Tetrazolium reductase and adrenaline oxidase in rat liver microsomalfractions Units of Vma.. and conditions are as indicated in Table 1 and 3. Kinetic data are obtained from double-reciprocal plots at the following substrate concentrations: Nitro Blue Tetrazolium, 1, 1.5, 2.5, 3.5, 5, 10 and 25.uM; NADPH, 100pM; adrenaline, 3, 5, 10, 20, 25, 50, 100 and 300pM; NADPH, 100pM; NADPH, 1, 1.5, 3, 6, 9, 19 and 30,M; Nitro Blue Tetrazolium, 100pUM; NADPH, 8 12, 20, 42, 84 and 126pM; adrenaline, 300p4M. Nitro Blue Tetrazolium reductase

Km for substrate (pM) Vmax. for substrate Km for NADPH (aM) Vmax. for NADPH





3.2 43.0 3.0 41.4

2.4 35.2 2.5

02 -dependent pathway 7.0 7.5 5.7



the addition of menadione causes activation of 02


dependent reactions and inhibition of the reduction of those acceptors which receive the electrons directly from the flavoprotein. Iyanagi & Yamazaki (1969) demonstrated that during one-electron transport from NADPH-cytochrome c reductase to menadione, a semiquinone form of menadione is created. From these investigations it may be concluded that acceleration of adrenaline oxidation and 02--dependent Nitro Blue Tetrazolium reduction after adding menadione is connected with the formation of 02-

in the process of menadione autoxidation. We have defined the kinetic constants for Nitro Blue Tetrazolium reduction and adrenaline oxidation by liver microsomal fractions. The data shown in Table 4 show that the Km for 0j'-dependent and 02--independent Nitro Blue Tetrazolium reduction hardly differ. Apparently as a consequence, Nitro Blue Tetrazolium is reduced by liver microsomal fractions in both pathways, but with other acceptors, in particular cytochrome c, which can be reduced through 02- by using the xanthine-xanthine oxidase system (McCord & Fridovich, 1969), the affinity for 02'-independent reduction is considerably stronger than for the 02-'-dependent one. This evidently explains why cytochrome c in liver microsomal fractions accepts electrons only directly from

NADPH-cytochrome c reductase. To oxidize adrenaline to adrenochrome at pH8.0 the participation of Vel. 154

Adrenaline oxidase 62.5 16.6 32.0


02-- becomes necessary (Misra & Fridovich, 1971). Hence, complete inhibition of this reaction by superoxide dismutase is observed in liver microsomal fractions. The stoicheiometry between NADPH oxidation and Nitro Blue Tetrazolium reduction in liver microsomal fractions defined in our experiments was 1:1 for both O2--dependent and 02--independent Nitro Blue Tetrazolium reductase. Discussion The results obtained in the present study show that at least two acceptors exist which react with O2 generated by the microsomal NADPH-specific flavoprotein, namely adrenaline and Nitro Blue Tetrazolium. The latter evokes especial interest since it is reduced both with the participation of 02- and by accepting electrons directly from microsomal flavoprotein. Measurement of the 02--dependent rate of interaction between the acceptors and microsomal fractions apparently allows one to draw some conclusions as to the rate of one-electron oxygen reduction by flavoprotein. Misra & Fridovich (1971) demonstrated that at pH7.8 in 0.05M-potassium phosphate buffer, in the presence of EDTA, 1.39mol of 02'- is required for the formation of 1 mol of adrenochrome from 1 mol of adrenaline. Under these conditions, rat liver



microsomal fractions oxidize adrenaline to adrenochrome at a velocity of 12.0nmol/min per mg of protein. On this basis it may be determined that the rate of 02 generation by liver microsomal fractions are 16nmol/mtnpermg ofprotein. On theother hand, it is known that the reduction ofone nolecule ofNitro Blue Tetrazolium re,quires two' .rediucing equivalents in the 02-independent pathway, whereas the 02--dependent pathway needs two superoxide anions. Then the rate.of 02- generation by liver microsomal fractions, determined from the 02-dependent rate of Nitro Blue Tetrazolium reduction (7.5 nmol/min per mg of protein) will be 15 nmol/min per mg o protein. According to Staudinger et al. (1965) the rate of oxygen uptake during acetanilide metabolism by liver microsomal fractions is 5-7nmol of 02/min per mg of protein. Depending on the metabolizing substrate the oxygen uptake is known to vary from 6 to 12nmol of 02/min per mg of protein (Staudt et al., 1974). Hence it follows that NADPH-specific flavoprotein in liver microsomal fractions reacts with oxygen at a rate comparable with the amount of oxygen consumed during xenobiotic metabolism reactions taking place with the participation of cytochrome P-450. References Aust, C. D., Roering, D. L. & Pederson, T. C. (1972) Biockem. Biophys. Res. Commwn. 47, 1133-1137 Fong, K. L., McCay, P. B., Poyer, J. L., KeJe, B. B. & Misra, H. (1973) J. Biol. Chem. 248, 7792-7797

Gnosspdlius, Y., Thor, H. & Orrenius* S. (1969) Chem.-

Biol. Interact. 1, 125-137 Gomall, A. G., Bardawill, C. 1. & David, M. M. (1949) J. Biol. Chem. 177, 751-766 Green, S., Masur, A. & Shorr, E. (1956)J. Biol. Chem. 220, 237-255 Iyanagi, T. & Yamazaki, I. (1969) Biochim. Biophys. Acta 172,370-381 Iyanagi, T., Makino, N. & Mason, H. S. (1974) Biochemistry 13, 1701-1710 Masters, B. S. S., -Kamin, H., Gibson, Q. H. & Williams, C. H. (1965) J. Biol. Chem. 248, 921-931 McCord, J. M. & Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055 Misra, H. P. & Fridovich, I. (1971) J. Biol. Chem. 246, 6886-6890 Pederson, T. C. & Aust, S. D. (1972) Biochem. Biophys. Res. Commun. 48,789-795 Philips, A. H. & Langdon, R. G. (1962)J. Biol. Chem. 237, 2652-2659 Prema Kumar, R., Ravindranath, S. D., Vaidyanathan, C. S. & Appaji Rao, N. (1972) Biochem. Biophys. Res. Commun. 49, 1422-1426 Roering, D. L., Mascaro, L. & Aust, S. D. (1972) Arch. Biochem. Biophys. 153, 475-479 Schatz, G. (1967) Methods Enymol. 10, 30-33 Staudinger, H., Kerekjarto, B., Ullrich, V. & Zubrycki, Z. (1965) in Oxidases and Related Redox Systems (King, T. E., Mason, H. S. & Morrison, M*, eds.), pp. 815-832, John Wiley and Sons, New York, London and Sydney Staudt, H., Lichtenberger, F. & Ulirich, V. (1974) Eur. J. Biochem. 46, 99-106 Steyn-Parv6, E. P. & Beinert, H. (1958)J. Bil. Chem. 233, 843-852 Tsyrlov, I. B., Mishin, V. M. & Lyakhovich, V. V. (1972) Life Sci. 11, 1045-1054


Interactions of some acceptors with superoxide anion radicals formed by the NADPH-specific flavoprotein in rat liver microsomal fractions.

307 Biochem. J. (1976) 154, 307-310 Printed in Great Britain Ineractions of Some Acceptors with Superoxide Anion Radicals Formned by the NADPPH-Spe...
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