scholarly journals Relationship between the reduction of oxygen, artificial acceptors and cytochrome P-450 by NADPH-cytochrome c reductase

1977 ◽  
Vol 168 (2) ◽  
pp. 133-139 ◽  
Author(s):  
V Lyakhovich ◽  
V Mishin ◽  
A Pokrovsky
Keyword(s):  
Cytochrome C ◽  
Microsomal Fraction ◽  
Metabolic Inhibitor ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Cytochrome C Reduction ◽  
Km Value ◽  
Liver Microsomal Fraction ◽  
Cytochrome P 450 ◽  
Nadph Cytochrome C Reductase

The interaction of NADPH—cytochrome c reductase with oxygen, artificial acceptors and cytochrome P-450 was studied. The generation of superoxide anion radicals (O2-.) from the oxidation of adrenaline to adrenochrome catalysed by NADPH—cytochrome c reductase proceeds independently of the interaction of the enzyme with the artificial anaerobic acceptors cytochrome c or 2,6-dichlorophenol-indophenol. Propyl 3,4,5-trihydroxybenzoate inhibited competitively the adrenaline oxidation by isolated NADPH—cytochrome c reductase (Ki 3.2—4.7 micrometer) and inhibited non-competitively the cytochrome c reduction (Ki 92—109 micrometer). In contrast with the process of electron transfer to cytochrome c, the rate of reduction of cytochrome P-450 and the rate of oxidation of adrenaline in liver microsomal fraction are correlated. Hexobarbital increases the Vmax. of adrenaline oxidation without affecting the Km value, whereas metyrapone, a metabolic inhibitor decreases Vmax. without affecting the Km. From the results obtained, some conclusions about NADPH—cytochrome c reductase function were made.

scholarly journals Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction

1986 ◽  
Vol 235 (2) ◽  
pp. 365-373 ◽  
Author(s):  
I Benveniste ◽  
B Gabriac ◽  
F Durst
Keyword(s):  
Cytochrome C ◽  
Microsomal Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Jerusalem Artichoke ◽  
Higher Plant ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Fixed Concentration ◽  
Cytochrome P 450 ◽  
Nadph Cytochrome C Reductase

NADPH-cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was solubilized by detergent from microsomal fraction of wounded Jerusalem-artichoke (Helianthus tuberosus L.) tubers and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2′,5′-bisphosphoadenosine-Sepharose 4B. An Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme exhibited typical flavoprotein redox spectra and contained equimolar quantities of FAD and FMN. The purified enzyme followed Michaelis-Menten kinetics with Km values of 20 microM for NADPH and 6.3 microM for cytochrome c. In contrast, with NADH as substrate this enzyme exhibited biphasic kinetics with Km values ranging from 46 microM to 54 mM. Substrate saturation curves as a function of NADPH at fixed concentration of cytochrome c are compatible with a sequential type of substrate-addition mechanism. The enzyme was able to reconstitute cinnamate 4-hydroxylase activity when associated with partially purified tuber cytochrome P-450 and dilauroyl phosphatidylcholine in the presence of NADPH. Rabbit antibodies directed against plant NADPH-cytochrome c reductase affected only weakly NADH-sustained reduction of cytochrome c, but inhibited strongly NADPH-cytochrome c reductase and NADPH- or NADH-dependent cinnamate hydroxylase activities from Jerusalem-artichoke microsomal fraction.

Temperature Influence on Basal Activity and Induction of Mixed Function Oxygenase Activity in Fundulus heteroclitus

1979 ◽  
Vol 36 (11) ◽  
pp. 1400-1405 ◽  
Author(s):  
John J. Stegeman
Keyword(s):  
Cytochrome C ◽  
Fundulus Heteroclitus ◽  
Reductase Activity ◽  
Control Fish ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Pyrene Hydroxylase ◽  
Mixed Function Oxygenase ◽  
Cytochrome P 450 ◽  
Nadph Cytochrome C Reductase

Treatment of Fundulus heteroclitus acclimated to 6.5 °C with benzo(a)pyrene did not elicit any change in the levels of hepatic microsomal NADH- or NADPH-cytochrome c reductase activity, nor in the levels of cytochrome P-450 or its catalytic activities. However, the same treatment offish at 16 5 °C resulted in a marked induction of benzo(a)pyrene hydroxylase and NADPH-cytochrome c reductase. Cytochrome P-450 content was also higher in the warm, treated fish and the Soret maximum of reduced, CO-treated microsomes was shifted to the violet. Levels of aminopyrine demethylase and NADH-cytochrome c reductase activities did not show a significant treatment effect. At neither temperature could treated and control fish be distinguished on the basis of in vitro inhibition of benzo(a)pyrene hydroxylase activity by 7,8-benzoflavone. Levels of NADPH-cytochrome c reductase and benzo(a)pyrene hydroxylase activities were greater in control Fundulus acclimated to 6.5 °C than to 16.5 °C, when normalized to microsomal protein, but not when based on body weight. The results indicate that habitat temperature alone may not affect the capacity for initial hydrocarbon metabolism in fish, but that it can strongly influence the induction of cytochrome P-450. Key words: temperature, cytochrome P-450, hydrocarbon metabolism, mixed-function oxygenase, Fundulus heteroclitus

scholarly journals Electron-transport pathway of the NADH-dependent haem oxygenase system of rat liver microsomal fraction induced by cobalt chloride

1979 ◽  
Vol 178 (2) ◽  
pp. 323-329 ◽  
Author(s):  
Y Hino ◽  
S Minakami
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Cytochrome B5 ◽  
Transport Pathway ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Haem Oxygenase ◽  
Reported Data ◽  
Nadph Cytochrome C Reductase

The hepatic microsomal haem oxygenase activity of rats treated with CoCl2 was studied kinetically by measuring biliverdin, the immediate product of the reaction. Biliverdin was extracted with diethyl ether/ethanol mixture, and was determined by the difference between A690 and A800. The apparent Km value for NADPH (at 50 microM-haematin) was about 0.2 microM when an NADPH-generating system was used, whereas that for NADH was about 630 microM. Essentially the same Vmax. values were obtained for both the NADH- and NADPH-dependent haem oxygenase reactions. No synergism was observed with NADH and NADPH. The NADH-dependent reaction was competitively inhibited by NADP+, with a Ki of about 10 microM. The inhibitoin of the NADH-dependent reaction by the antibody against rat liver microsomal NADPH-cytochrome c reductase was essentially complete, with a pattern similar to that of the NADPH-dependent reaction. The immunochemical experiment and the comparison of the kinetic values with the reported data on isolated NADH-cytochrome b5 reductase and NADPH–cytochrome c reductase indicated the involvement of the latter enzyme in NADH-dependent haem oxygenation by microsomal fraction in situ.

Release of ferrous iron from ferritin by liver microsomes: a possible role in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin

1984 ◽  
Vol 62 (12) ◽  
pp. 1293-1300 ◽  
Author(s):  
Bruce Rowley ◽  
George D. Sweeney
Keyword(s):  
Cytochrome C ◽  
Flavin Mononucleotide ◽  
Monooxygenase System ◽  
Iron Release ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Ferritin Iron ◽  
Tetrachlorodibenzo P Dioxin ◽  
Cytochrome P 450 ◽  
Nadph Cytochrome C Reductase

Nonheme iron is synergistic with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in producing hepatotoxicity in mice. Fe2+ rather than Fe3+ is the probable toxin and we speculated that TCDD, an inducer of microsomal electron transport, might favour reduction of iron. We have defined a system which will release Fe2+ from ferritin (Fe3+) under anaerobic conditions and in the presence of added flavin mononucleotide (FMN). The rate of reduction of ferritin iron was proportional (a) to microsomal protein from 0.5 to >3 mg/mL, (b) to the activity of NADPH–cytochrome c reductase over 0.1 U/mL, (c) to ferritin at concentrations exceeding iron concentrations >200 μmol/L, and (d) to the concentration of FMN when it was less than 125 μmol/L. The system was approximately twice as active with NADPH as with NADH as electron donor. The linear phase of iron release did not commence immediately, but followed a delay (±0.5 min) after adding FMN to an anaerobic mixture containing microsomes, ferritin, an NADPH-generating system, and an oxygen-scavenging system. When microsomes from untreated, Phenobarbitaltreated (3 days), or TCDD-treated (1 or 3 weeks) rats were compared, iron release correlated most closely with the cytochrome P-450 concentration. However, when the microsomal proteins were solubilized and the NADPH–cytochrome c reductase and cytochrome P-450 activities were separated, reduction of ferritin iron was shown to be a function only of the reductase fraction, except that the delay in initiating release of Fe2+ was increased with purified reductase and decreased when a monooxygenase system was reconstituted with cytochrome (phenobarbital or TCDD induced) and lipid. These studies have defined a potentially important hepatic microsomal system able to release Fe2+ from ferritin iron, but have failed to indicate any feature unique to the dioxin-induced monooxygenase system.

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