John B. Schenkman

The many roles of cytochrome b5.

Four distinct suggestions have been made to explain the mechanism of the cytochrome b(5)-imposed positive modifier action of the cytochrome P450 monooxygenase reaction. The first mechanism involves a direct input of an electron into the monooxygenase cycle. This is the second of the two electrons necessary for activation of molecular oxygen, and appears to be a rate-limiting step in the monooxygenase reaction. P450 monooxygenases all appear to be uncoupled to varying extents, releasing superoxide and hydrogen peroxide instead of oxidized substrate. A second mechanism suggests that cytochrome b(5) acts as a positive modifier of the monooxygenase by decreasing the extent of uncoupling of the monooxygenase reaction. The implication is that a slow input of the second electron allows uncoupling of a superoxide anion instead of formation of two-electron reduced oxygen. Faster input of the second electron via cytochrome b(5) would result in formation of more of the activated oxygen that reacts with substrate to form product. A third suggestion involves formation of a two-hemoprotein complex between cytochrome b(5) and cytochrome P450 that allows acceptance of two electrons from NADPH-cytochrome P450 reductase. Uncomplexed cytochrome P450 accepts an electron from the reductase, dissociates from it, binds oxygen, and re-associates with the reductase to accept another electron. Complexation with cytochrome b(5) enhances the rate of formation of the active oxygen by obviating the need for two interactions with reductase. The fourth mechanism has cytochrome b(5) serving as an effector without a reduction-oxidation role in the monooxygenation reaction. This effector function may be to enhance the breakdown of the oxygenated hemoprotein to products or to facilitate flow of electrons through the system.

Kinetic parameters of drug-metabolizing enzymes in Ca2+-sedimented microsomes from rat liver☆

Abstract Kinetic parameters of five substrates of the mixed function oxidase system were determined to ensure that the mixed function oxidases were unaltered by the Ca 2+ -sedimented microsomes. Using either of the microsomal preparation procedures, no differences were noted in the V max and K m for aminopyrine, ethylmorphine and p -nitroanisole demethylation, aniline hydroxylation and hexobarbital oxidase. Also, no differences were seen in the NADPH cytochrome P-450 reductase activities. The Ca 2+ -sedimentation procedure was further simplified to allow microsomal preparation within 1 hr.

[6] Preparation of microsomes with calcium

Publisher Summary This chapter focuses on the preparation of microsomes with calcium. Isolation of the microsomal fraction of the hepatic cell employs a procedure of differential ultracentrifugation. This method is developed for mammalian liver tissue, but also employed to obtain microsomal fractions from a variety of tissues, such as lung, kidney, spleen, adrenals. Other procedures used to isolate microsomes are developed to replace ultracentrifugation for two main reasons, (1) differential ultracentrifugation requires an expensive ultracentrifuge; (2) it is time consuming, requiring 2 hr of ultracentrifugation. Two methods that are used to isolate microsomes from the postmitochondrial fraction are acid precipitation and gel filtration. In recent years, one of the procedures for microsomal isolation that is extensively studied and gaining acceptance in mammalian liver studies is a method involving aggregation of microsomes with calcium. Basically, this involves the addition of Ca 2+ ions to the postmitochondrial supernatant followed by a short centrifugation at speeds obtainable with most refrigerated centrifuges, such as the Sorvall RC-2B.

Comparative expression profiling of 40 mouse cytochrome P450 genes in embryonic and adult tissues

This study is the first systematic investigation of the gestational age-dependent and adult tissue-specific expression patterns of each known mouse CYP family (40 genes) using normalized cDNA panels and uniform reverse transcriptase polymerase chain reaction-based assays. Twenty-seven of the P450s were constitutively expressed during development. The number gradually increased through the phases of gastrulation E7 (n=14), neural patterning and somitogenesis E11 (n=17), organogenesis E15 (n=20), and fetal period E17 (n=21). Cyp2s1, Cyp8a1, Cyp20, Cyp21a1, Cyp26a1, Cyp46, and Cyp51 were detected in each of the four stages studied. Members of family CYP1 demonstrated complex, nonoverlapping embryonic patterns of expression, indicating that Cyp1a1 and Cyp1a2 may not compensate for Cyp1b1 deficiency associated with abnormal eye development. Multiple Cyp forms were found to be constitutively expressed in each of the adult tissues studied: liver (n=31), kidney (n=30), testis (n=26), lung (n=24), and heart (n=13). The tissue-specific P450-expression profiles reported in this study provide a reference for more focused analysis of the tissue-specific and developmental functions of the cytochrome P450 monooxygenases.

Mechanism of cholestasis: III. Interaction of synthetic detergents with the microsomal cytochrome P-450 dependent biotransformation system in vitro A comparison between the effects of detergents, the effects of bile acids, and the findings in bile duct ligated rats

Abstract Synthetic nonionic and anionic detergents interact with the cytochrome P-450 dependent microsomal biotransformation system in three somewhat overlapping stages depending on dose and time of exposure. In the first stage, the detergents bind to P-450, presumably to a lipoprotein region (binding site I). This is reflected in a spectral change of P-450, the enhancement of the P-450 reductase, and the competitive inhibition of the metabolism of other Type I substrates, like aminopyrine. In the second stage the mixed inhibition of the aminopyrine demethylation without a proportional loss of P-450 suggests a blocking or solubilization of the lipoprotein binding site. In the third stage, degradation of P-450 and solubilization of P-450 reductase reflects the breakdown of the enzyme system. These stages correspond to the effect on microsomes of dihydroxy bile acids in vitro and to some degree of bile duct ligation in vivo . These observations suggest a detergent effect of bile acids as an explanation of the alterations of the endoplasmic reticulum in cholestasis.

[109] Methods for the elevation of hepatic microsomal mixed function oxidase levels and cytochrome P-450

Publisher Summary The chapter describes the methods for the elevation of hepatic microsomal mixed function oxidase levels and cytochrome P-450. Many substances have been shown to elevate the content of the mixed function oxidase activity of liver microsomes; these range from barbiturates to tranquilizers, insecticides, and polycyclic hydrocarbons such as 3,4-benzpyrene and 3-methylcholanthrene. The increase in oxidative activity is associated with an increase in the microsomal content of cytochrome P-450. The time necessary to achieve maximal levels of microsomal mixed function oxidase activities varies with the inducer compound used. Methylcholanthrene or benzpyrene treatment exerts a maximal effect within 24 hours. After one injection of DDT, the maximal activity is achieved after 1 or 2 weeks. When phenobarbital is used, maximal enzyme activity toward all substrates is reached after 3-5 days of treatment. Three procedures, which increase the level of liver microsomal mixed function oxidase activity and cytochrome P-450, are described. One method uses Phenobarbital as the inducer; a second uses 3-methyleholanthrene or 3,4-benzpyrene; and the third method employs DDT. The chapter also discusses the preparation of microsomes.

Direct electron injection from electrodes to cytochromeP450cam in biomembrane-like films

Direct, reversible, electron transfer has been demonstrated between electrodes and native cytochrome P450 cam (cyt P450 cam ) in lipid films. Electron injection directly from electrodes into the haem Fe III of cyt P450 cam has been confirmed by the influence of the known reaction of cyt P450 cam Fe II with CO on the voltammetric midpoint potential. Analysis of square wave voltammograms suggested a distribution of enzyme formal potentials in the films. With oxygen present, more than one electron was injected into the enzyme in films, mimicking in vivo electron acceptance by cyt P450Fe III and cyt P450Fe II –O 2 during catalytic oxidations. Cyt P450 cam films also catalysed electrochemically driven reduction of trichloroacetic acid in anaerobic solutions. These stable enzyme–lipid films may find applications in fundamental biochemical and toxicity studies, biocatalysis and biosensors.

The influence of phenobarbital on the turnover of hepatic microsomal cytochrome b5 and cytochrome P-450 hemes in the rat.

Abstract The turnover of 14 C-labeled heme in steady state phenobarbital-induced rats was compared with the turnover of the same hemes in control rats. The half-lives of cytochrome b 5 and cytochrome P-450 hemes in the steady-state induced animals were unchanged (45 and 22 h, respectively). Induction of cytochrome P-450 was found to be caused by an increased rate of synthesis, measured by the rate of δ-amino[4- 14 C]levulinate incorporation into heme in the early stages of phenobarbital treatment. When the level of hemoproteins during phenobarbital administration had reached a steady-state induced level, the rate of home destruction was elevated to balance the induced rate of synthesis.

Diethylaminoethyl 2,2-diphenylvalerate HCl (SKF 525-A)—In vivo and in vitro effects of metabolism by rat liver microsomes—Formation of an oxygenated complex

Abstract Metabolism of diethylaminoethyl 2,2-diphenylvalerate HCl (SKF 525-A) or its primary amine analogue causes formation of a stable oxygenated complex of ferrous P-450. The complex is also generated in vivo and survives preparation of liver microsomes. Formation of the complex requires active metabolism of either SKF 525-A or the N -dealkylated primary amine, SKF 26754A. This new species has an absorption maximum at 455 nm. Oxidation of the hemoprotein with potassium ferricyanide causes loss of the absorption band, but subsequent reduction and oxygenation of the medium restore it. This new complex is believed to be responsible for the observed noncompetitive inhibition of drug metabolism in vitro by SKF 525-A.

Spin state control of the hepatic cytochrome P450 redox potential.

Abstract We have measured the oxidation-reduction potential of isolated and partially purified cytochrome P450 from uninduced rat liver, both in the presence and absence of Type I substrates. Native P450 is found to have a potential of −300 mV with respect to the standard hydrogen electrode, while the addition of benzphetamine or hexabarbital increases the redox potential to −237 mV and −225 mV respectively. Quantitation of the thermally induced S = 1 2 to S = 5 2 spin transition of the ferric heme iron for this P450 preparation both substrate free as well as with bound benzphetamine and hexabarbital indicate an increase in the high spin ( S = 5 2 ) fraction on the binding of Type I substrates. The relevant oxidation-reduction equilibria of the heme chromaphore are presented in terms of a thermodynamic model for the control of the observed cytochrome P450 redox potential through this modulation of the spin configuration of the five d-electrons of the ferric heme iron induced by the binding of Type I substrates.