Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver

Wang, Philip; Mason, P S; Guengerich, F. Peter

doi:10.1016/0003-9861(80)90274-x

Cited by 86 publications

(50 citation statements)

References 41 publications

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“…Our values were similar to those found for biopsies [9,13,24,, but the cytochrome P-450 content was higher than in microsomes prepared from autopsy samples [18,20]. Cytochrome P-450 and epoxide hydrolase content was lower in human than in rat livers.…”

Section: Enzyme Contentsupporting

confidence: 88%

Cytochrome P-450 Monooxygenase Activities in Human and Rat Liver Microsomes

et al. 1981

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Microsomes were prepared from human livers obtained from renal donors of various ages and both sexes. Their drug-metabolizing capacity was measured and compared to that of rat liver microsomes. The following parameters were investigated: cytochrome P-450, cytochrome bs, NADPH -cytochrome c reductase, epoxide hydrolase, aryl hydrocarbon hydroxylase, benzphetamine N-demethylase, p-nitroanisole-0-demethylase, ethoxycoumarin-0-deethylase, steroid-16a-hydroxylase. In addition, the metabolism of benzo(a)pyrene, progesterone, pregnenolone, testosterone, dehydroepiandrosterone and estradiol was studied in detail in vitro. The inhibitory effect of metyrapone and a-naphthoflavone on 7-ethoxycoumarin-0-deethylase was measured. The microsomal proteins of both species were separated by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate.The following conclusions were drawn from the results obtained. Human liver microsomes can be stored under optimal conditions for the measurement of a large variety of enzymic activities. Human liver microsomes are able to metabolize the various xenobiotics used as substrates with a rate similar to that of female rat liver microsomes. No sex-linked difference in enzymic activity was observed in human microsomes. Significant differences in benzo(a)pyrene and steroid metabolism were registered when human and rat liver microsomes were compared. The monooxygenase activities, the sensitivity to in v i m a-naphthoflavone and metyrapone, the results of steroid metabolism, and slab gel electrophoresis are strong indications for multiplicity of human liver cytochrome P-450.Without appropriate means, living organisms would never be able to eliminate from their systems the lipophilic compounds which are either produced by their own metabolism (i.e. steroid hormones, fatty acids, prostaglandins), or are accidentally or voluntarily absorbed from their environment (i.e. food additives, drugs, pesticides, pollutants . . .). Living organisms have thus developed a number of enzymic systems which transform these substances into more polar and therefore more hydrophilic metabolites which can be easily excreted via the urine or feces.The first step in these metabolic transformations is usually catalyzed by (cytochrome P-450)-dependent monooxygenases. These microsomal multienzyme complexes oxidize a great variety of endogenous as well as exogenous substrates. Their biochemical and biological properties have been extensively studied in laboratory animals [l -41. The different types of cytochrome P-450 are characterized by their substrate specificity. Their qualitative and quantitative proportions in a given tissue vary largely as a function of physiological, pharmacological and pathological parameters [2,3,5].Trivia! Numes. r-Naphthoflavone, 7,8-benzoflavone; rnetyrapone, 2-methyl-1,2-di-3-pyridyl-l -propanone; progesterone, pregn-4-en-3,20-dione; pregnenolone, 3p-hydroxypregn-5-en-20-one; testosterone, androst-4-en-I7f1'-01-3-one; dehydroepiandrosterone, 3p-hydroxyandrost-5-en-1 7-one; estradiol...

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Section: Enzyme Contentsupporting

confidence: 88%

Cytochrome P-450 Monooxygenase Activities in Human and Rat Liver Microsomes

et al. 1981

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“…obtained from sources as described previously or were of the highest quality commercially available (Wang et al, 1980;Shimada et al, 1994Shimada et al, , 2002aShimada et al, , 2002bMiyazawa et al, 2002a,b). Enzymes and Antibodies.…”

Section: Methodsmentioning

confidence: 99%

Roles of Human Cyp2a6 and 2b6 and Rat Cyp2c11 and 2b1 in the 10-Hydroxylation of (–)-Verbenone by Liver Microsomes

Miyazawa¹,

Sugie²,

Shimada³

2003

Drug Metab Dispos

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:(؊)-Verbenone, a monoterpene bicyclic ketone, is a component of the essential oil from rosemary species such as Rosmarinus officinalis L., Verbena triphylla, and Eucalyptus globulus and is used for an herb tea, a spice, and a perfume. In this study, (؊)-verbenone was found to be converted to 10-hydroxyverbenone by rat and human liver microsomal cytochrome P450 (P450) enzymes. The product formation was determined by high-performance liquid chromatography with UV detection at 251 nm. There was a good correlation between activities of coumarin 7-hydroxylation and ( hydroxylation at all, suggesting that there were species-related differences in the catalytic properties of human and rat CYP2A enzymes in the metabolism of (؊)-verbenone. In the rat, recombinant CYP2C11, CYP2B1, and CYP3A2 catalyzed (؊)-verbenone 10-hydroxylation with V max and K m ratios (ml/min/nmol P450) of 0.73, 0.20, and 0.03, respectively. Male-specific CYP2C11 was a major enzyme in (؊)-verbenone 10-hydroxylation by untreated rat livers, and CYP2B1 catalyzed this reaction in liver microsomes of phenobarbital-treated rats. Rat CYP2C12, a female-specific enzyme, did not catalyze (؊)-verbenone 10-hydroxylation. These results suggest that human CYP2A6 and rat CYP2C11 are the major catalysts in the metabolism of (؊)-verbenone by liver microsomes and that there are speciesrelated differences in human and rat CYP2A enzymes and sex-related differences in male and female rats in the metabolism of (؊)-verbenone.

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“…Earlier reports had observed that isoforms of human cytochrome P-450 could be recon stituted with human, rat [9], rabbit, porcine, and bovine NADPH-cytochrome P-450 re ductase [10], and since the preparation of spe cies-specific reductase requires additional pu rification steps [11], it seemed justified to use rat NADPH-cytochrome P-450 reductase in reconstitution systems with interspecific forms of cytochrome P-450. However, the fact that rat NADPH-cytochrome P-450 re ductase can be reconstituted with cyto chromes P-450 isolated from other species does not mean that it is as equally effective as the conspecific reductases.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Isolation of NADPH-Cytochrome P-450 Reductase and Cytochrome P-450 Using Tentacle Ion-Exchange Chromatography and Interspecies Comparison of the Reductase Activity

Sharma¹,

Jeong²,

Sharma³

et al. 1995

Pharmacology

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Using the same initial Fractogel (tentacle) ion-exchange chromatography to isolate murine cytochrome P-450, mouse hepatic NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was simultaneously isolated from solubilized liver microsomes and purified on a DE-52 column to a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 77 kD, and its specific activity was 25.4 µmol·min^–1·mg protein^-1. Purified constitutive mouse liver NADPH-cytochrome P-450 reductase was successfully reconstituted in vitro with dilauroylphosphatidyl-choline and constitutive purified mouse testosterone 2α-hydroxylase (cytochrome P-450_2α) with an observed activity of 13.8 nmol·min^–1·nmol P-450^–1. Although the partially purified reductase obtained from the Fractogel column was contaminated by significant levels of two unidentified proteins, it was as equally effective in the reconstituted system as the DE-52-derived purified reductase. Lastly, we found that rat and mouse NADPH-cytochrome P-450 reductases were similarly effective in supporting the catalytic activity of rat cytochrome P-450 2B1, but the murine reductase was 50% more effective than the rat reductase in a reconstituted system containing mouse cytochrome P-450_2α·

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Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver

Cited by 86 publications

References 41 publications

Cytochrome P-450 Monooxygenase Activities in Human and Rat Liver Microsomes

Cytochrome P-450 Monooxygenase Activities in Human and Rat Liver Microsomes

Roles of Human Cyp2a6 and 2b6 and Rat Cyp2c11 and 2b1 in the 10-Hydroxylation of (–)-Verbenone by Liver Microsomes

Simultaneous Isolation of NADPH-Cytochrome P-450 Reductase and Cytochrome P-450 Using Tentacle Ion-Exchange Chromatography and Interspecies Comparison of the Reductase Activity

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