Properties of partially purified liver microsomal cytochrome <i>P</i>‐450: Acceptance of two electrons during anaerobic titration

Ballou, David P.; Veeger, C.; Hoeven, Theodore A. van der; Coon, Minor J.

doi:10.1016/0014-5793(74)80086-4

Cited by 33 publications

(5 citation statements)

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“…The kinetic data presented in Figures 3 and 4 indicate that under our assay conditions the Km value foi NADPH is 5 /uM, whether cytochrome P-450 or cytochrome c is used as the electron acceptor, and raise the question of the mechanism of reduction of the enzyme by NADPH. This question is of particular interest in view of the recent report that cytochrome P-450, the physiological electron acceptor for cytochrome P-450 reductase, requires 2 reducing equivalents for complete reduction (Ballou et a!., 1974). The aerobic re duction by NADPH of cytochrome c reductase, the proteaseor lipase-solubilized microsomal flavoprotein, has been studied extensively by Masters et al (1965a,b), Iyanagi and Mason (1973), Iyanagi et al (1974), and Masters et al (1975).…”

Section: Resultsmentioning

confidence: 99%

“…The lipid component was identified as phosphatidylcholine (Strobe1 et al, 1970), and cytochrome P-450 has been purified to homogeneity (van der Hoeven et ai., 1974;Ryan et al, 1975a). Characterization of the oxidation-reduction properties (Ballou et al, 1974;Guengerich et al, 1975) and the existence of multiple forms of cytochrome P-450 (Ryan et al, 1975b;Haugen et al, 1975) have also been reported. Early efforts to solubilize the reductase with proteolytic (Phillips and Langdon, 1962;Pederson et al, 1973) or lipolytic (Williams and Kamin, 1962) enzymes yielded a flavoprotein capable of reducing electron acceptors such as cytochrome c but unable to support cytochrome P-450 dependent substrate hydroxylation (Masters et al, 1975).…”

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confidence: 99%

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NADPH-cytochrome P-450 reductase from rat liver: purification by affinity chromatography and characterization

Dignam¹,

Strobel²

1977

Biochemistry

209

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(NADPH)-cytochrome P-450 reductase was purified to apparent homogeneity by a procedure utilizing nicotinamide adenine dinucleotide phosphate (NADP)-Sepharose affinity column chromatography. The purified flavoprotein has a molecular weight of 79 700 and catalyzes cytochrome P-450 dependent drug metabolism, as well as reduction of exogenous electron acceptors. Aerobic titration of cytochrome P-450 reductase with NADPH indicates that an air-stable reduced form of the enzyme is generated by the addition of 0.5 mol of NADPH per mole of flavin, as judged by spectral characteristics. Further addition of NADPH causes no other changes in the absorbance spectrum. A Km value for NADPH of 5 micron was observed when either cytochrome P-450 or cytochrome c was employed as electron acceptor. A Km value of 8 +/- 2 micron was determined for cytochrome c and a Km of 0.09 +/- 0.01 micron was estimated for cytochrome P-450.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

NADPH-cytochrome P-450 reductase from rat liver: purification by affinity chromatography and characterization

Dignam¹,

Strobel²

1977

Biochemistry

209

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“…Cytochromes P450 comprise a family of hemoproteins which catalyze monooxidation of exogenous chemicals as well as endogenous compounds. During the monooxidation reaction, two electrons are transferred from NADPH to the P450 1 via P450 reductase (Ballou et al, 1974;Guengerich et al, 1975) and molecular oxygen is bound to the cytochrome P450 to form a oxyferro complex (Estabrook et al, 1971). Electron transfer to the oxygen results in production of an "activated oxygen" which is able to attack the substrate.…”

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confidence: 99%

Kinetics of Substrate Reaction in the Course of Hydroperoxide-Mediated Inactivation of Cytochrome P450 1A1

Liang²,

Strobel³

1996

Biochemistry

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A schematic kinetic model is proposed for the hydroperoxide-mediated substrate reaction and cytochrome P450 inactivation. From the model, the relationships between the product concentration at infinite time (P infinity), the apparent rate constant of P450 inactivation (A), and the substrate concentrations are predicted, and the predictions were experimentally examined. The reciprocal of P infinity is proportional to the reciprocal of the substrate concentration in both CuOOH- and H2O2-supported substrate reactions. The reciprocal of P infinity is proportional to cumene hydroperoxide (CuOOH) concentration, but P infinity is independent of H2O2 concentration, indicating different effects of CuOOH and H2O2 in P450 inactivation. The apparent rate constant (A) is proportional to the reciprocal of the substrate concentrations, suggesting substrate protection of P450 from hydroperoxide inactivation. The model also suggests that a second CuOOH molecule may compete with substrate for binding to the P450 substrate binding site. Simulated kinetics of production formation vs time are quite consistent with the experimental kinetics.

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“…Under anaerobic conditions two electrons are transferred from NADPH to the P-450,,-substrate complex via the reductase, one to the iron atom and the other to an unidentified acceptor (10,11). Molecular oxygen is bound to the ferrocytochrome to form a ternary complex, as shown by Estabrook et al (12) with liver microsomal suspensions and by Gunsalus et al (13) and by Ishimura et al (14) with bacterial cytochrome P-450.…”

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confidence: 99%

Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450

Nordblom

White

Coon

1976

Archives of Biochemistry and Biophysics

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349

112

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Highly purified liver microsomal cytochrome P-450 catalyzes the hydroperoxide-dependent hydroxylation of a variety of substrates in the absence of NADPH, NADPHcytochrome P-450 reductase, and molecular oxygen. The addition of phosphatidylcholine is necessary for maximal activity. The absence of flavoproteins and cytochrome b, from the cytochromeP-450 preparations rules out the involvement of other known microsomal electron carriers. The ferrous form of cytochrome P-450 is not involved in peroxidedependent hydroxylation reactions, as indicated by the lack of inhibition by carbon monoxide. With cumene hydroperoxide present, a variety of substrates is attacked, including N-methylaniline, N,N-dimethylaniline, cyclohexane, benzphetamine, and aminopyrine.With benzphetamine as the substrate, cumene hydroperoxide may be replaced by other peroxides, including hydrogen peroxide, or by peracids or sodium chlorite. A study of the stoichiometry indicated that equimolar amounts ofN-methylaniline, formaldehyde, and cumyl alcohol (o,o-dimethylbenzyl alcohol) are formed in the reaction of N,N-dimethylaniline with cumene hydroperoxide. Since H,180 is incorporated only slightly into cyclohexanol in the reaction of cyclohexane with cumene hydroperoxide, it appears that the oxygen atom in cyclohexanol is derived primarily from the peroxide. The data obtained are in accord with a peroxidase-like mechanism for the action of cytochrome P-450.The mixed function oxidase of liver microsomal membranes is capable of hydroxylating or otherwise metabolizing fatty acids and steroids as well as a variety of foreign compounds, including drugs, insecticides, alkanes, anesthetics, and carcinogens. This enzyme system was resolved into three components, P-450LM ,3 NADPHcytochrome P-450 reductase, and phosphatidylcholine (3-61, and more recently our

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Properties of partially purified liver microsomal cytochrome P‐450: Acceptance of two electrons during anaerobic titration

Cited by 33 publications

References 23 publications

NADPH-cytochrome P-450 reductase from rat liver: purification by affinity chromatography and characterization

NADPH-cytochrome P-450 reductase from rat liver: purification by affinity chromatography and characterization

Kinetics of Substrate Reaction in the Course of Hydroperoxide-Mediated Inactivation of Cytochrome P450 1A1

Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450

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