The High-Level Expression in Escherichia coli of the Membrane-Bound Form of Human and Rat Cytochrome b5 and Studies on Their Mechanism of Function

Holmans, P L; Shet, Manjunath S.; Martin-Wixtrom, Cheryl; Fisher, Charles; Estabrook, Ronald W.

doi:10.1006/abbi.1994.1345

Cited by 95 publications

(76 citation statements)

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“…For confirmation, cyto-b reductase activity was also determined in a 100-l reaction mixture containing 10 mM phosphate buffer at pH 6.6, 100 M NADH, 2 M cytochrome b 5 , and 10 g of microsome. Reduction of cytochrome b 5 was monitored at 427 nm for 10 min as described previously (Yubisui and Takeshita, 1982;Holmans et al, 1994). Purified rat P450 reductase was analyzed for its ability to reduce ferricyanide in the same manner with NADPH or NADH as an electron donor.…”

Section: Methodsmentioning

confidence: 99%

NAD(P)H:Quinone Oxidoreductase-1-Dependent and -Independent Cytotoxicity of Potent Quinone Cdc25 Phosphatase Inhibitors

Han¹,

Shen²,

Carr³

et al. 2004

J Pharmacol Exp Ther

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Cdc25 dual-specificity phosphatases coordinate cell cycle progression and cellular signaling. Consequently, Cdc25 inhibitors represent potential anticancer agents. We evaluated Ͼ10,000 compounds for inhibition of human Cdc25 phosphatases and identified many potent and selective inhibitors, which all contained a quinone. Bioreductive enzymes frequently detoxify or activate quinones. Therefore, we evaluated the effect of NAD(P)H:quinone oxidoreductase-1 (NQO1) and reductase-rich microsomes on the activity of three quinone-containing Cdc25 inhibitors: 2-(2-hydroxyethylsulfanyl)-3-methyl-1,4-naphthoquinone (Cpd 5, compound 5; NSC 672121), 2,3-bis-(2-hydroxyethylsulfanyl)-1,4-naphthoquinone (NSC 95397), and 6-chloro-7-(2-morpholin-4-ylethylamino)quinoline-5,8-dione (NSC 663284). Each inhibitor was reduced by human NQO1 (K m of 0.3-0.5 M) but none by microsomes. Compounds were evaluated with six cancer cell lines containing different amounts of NQO1: HT-29 (1056 nmol/mg/ min), HCT116 (660 nmol/mg/min), sublines HCT116-R30A (28 nmol/mg/min) and HCT-116R30A/NQ5 (934 nmol/mg/min), MDA-MB-231/Q2 (null NQO1), and subline MDA-MB-231/Q6 (124 nmol/mg/min) but containing similar amounts of microsomal cytochrome P450 reductase and cytochrome b 5 reductase. Growth inhibition and G2/M arrest by Cpd 5 was proportional to NQO1 levels, requiring 4-to 5-fold more Cpd 5 to inhibit HCT-116 or HCT-116R30A/NQ5 compared with HCT-116R30A. In contrast, in all tested cell lines irrespective of NQO1 level, growth inhibition and G2/M arrest by NSC 95375 and NSC 663284 were similar (average IC 50 of 1.3 Ϯ 0.3 and 2.6 Ϯ 0.4 M, respectively). NSC 95375 and NSC 663284 also caused similar Cdk1 hyperphosphorylation, indicating similar Cdc25 inhibition. However, lower Cpd 5 concentrations were needed to produce Cdk1 hyperphosphorylation in sublines with minimal NQO1. Thus, NQO1 detoxified Cpd 5, probably by reducing it to a less active hydroquinone, whereas NSC 95397-and NSC 663284-generated cytotoxicity was unaffected by NQO1.

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Section: Methodsmentioning

confidence: 99%

NAD(P)H:Quinone Oxidoreductase-1-Dependent and -Independent Cytotoxicity of Potent Quinone Cdc25 Phosphatase Inhibitors

Han¹,

Shen²,

Carr³

et al. 2004

J Pharmacol Exp Ther

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“…The assay of purified P450s requires that they be reconstituted with NADPH/cytochrome P450 reductase in a complex mixture that includes detergent, phospholipids, and reduced glutathione (Gillam et al, 1995). Some in vitro reconstitution experiments have shown that for a number of P450s, the inclusion of cytochrome b 5 can significantly increase substrate turnover by the monooxyenase system by improving the coupling between the cytochromes P450 and NADPH/cytochrome P450 reductase (Gorsky and Coon, 1986;Holmans et al, 1994;Bell and Guengerich, 1997). Cytochrome b 5 is a heme protein whose mechanism of action in reconstituted systems is not clear.…”

Section: Tablementioning

confidence: 99%

Stereo- And Regioselectivity Account for the Diversity of Dehydroepiandrosterone (Dhea) Metabolites Produced by Liver Microsomal Cytochromes P450

Miller

Cai²,

Ripp³

et al. 2004

Drug Metab Dispos

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:The purpose of this study was to quantify the oxidative metabolism of dehydroepiandrosterone (3␤-hydroxy-androst-5-ene-17-one; DHEA) by liver microsomal fractions from various species and identify the cytochrome P450 (P450) enzymes responsible for production of individual hydroxylated DHEA metabolites. A gas chromatography-mass spectrometry method was developed for identification and quantification of DHEA metabolites. 7␣-Hydroxy-DHEA was the major oxidative metabolite formed by rat (4.6 nmol/min/mg), hamster (7.4 nmol/ min/mg), and pig (0.70 nmol/min/mg) liver microsomal fractions. 16␣-Hydroxy-DHEA was the next most prevalent metabolite formed by rat (2.6 nmol/min/mg), hamster (0.26 nmol/min/mg), and pig (0.16 nmol/ min/mg). Several unidentified metabolites were formed by hamster liver microsomes, and androstenedione was produced only by pig microsomes. Liver microsomal fractions from one human demonstrated that DHEA was oxidatively metabolized at a total rate of 7.8 nmol/min/mg, forming 7␣-hydroxy-DHEA, 16␣-hydroxy-DHEA, and a previously unidentified hydroxylated metabolite, 7␤-hydroxy-DHEA. Other human microsomal fractions exhibited much lower rates of metabolism, but with similar metabolite profiles. Recombinant P450s were used to identify the cytochrome P450s responsible for DHEA metabolism in the rat and human. CYP3A4 and CYP3A5 were the cytochromes P450 responsible for production of 7␣-hydroxy-DHEA, 7␤-hydroxy-DHEA, and 16␣-hydroxy-DHEA in adult liver microsomes, whereas the fetal/neonatal form CYP3A7 produced 16␣-hydroxy and 7␤-hydroxy-DHEA. CYP3A23 uniquely formed 7␣-hydroxy-DHEA, whereas other P450s, CYP2B1, CYP2C11, and CYP2D1, were responsible for 16␣-hydroxy-DHEA metabolite production in rat liver microsomal fractions. These results indicate that the stereo-and regioselectivity of hydroxylation by different P450s account for the diverse DHEA metabolites formed among various species. Dehydroepiandrosterone(3␤-hydroxy-androst-5-ene-17-one; DHEA 2 ) is a 19-carbon steroid derived from cholesterol by a series of cytochrome P450 monooxygenase-and hydroxysteroid dehydrogenase-dependent reactions (Conley and Bird, 1997). In its sulfated form, DHEA is the most abundant circulating steroid in humans and is a precursor to the sex steroids, estrogen and testosterone. Levels of DHEA-S in the circulation are high during fetal development (1-5 M) but fall rapidly after birth and remain low for the first 5 years of life. DHEA and DHEA-S levels in blood then rise and peak during the second decade (ϳ10 M), followed by an age-dependent decline for individuals aged 30 or above (Herbert, 1995). The developmental changes in circulating levels of DHEA and DHEA-S in the blood are not paralleled by other steroid hormones, suggesting that the mechanisms regulating DHEA formation in adrenal are unique (Rainey et al., 2002). In contrast, serum cholesterol levels tend to increase with age, whereas DHEA levels decline with age. The decline in circulating ...

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“…Enzyme was quantitated using the reduced carbon monoxide difference spectrum as described previously (Schenkman and Jansson, 2006). Rat NADPH P450 oxidoreductase (Shen et al, 1989) and rat cytochrome b 5 (Holmans et al, 1994) were expressed and purified as described previously.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of Inhibition Selectivity for Human Cytochrome P450 2A Enzymes

2012

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ABSTRACT:Cytochrome P450 (P450) enzymes are mixed-function oxidases that catalyze the metabolism of xenobiotics and endogenous biochemicals. Selective inhibitors are needed to accurately distinguish the contributions of individual P450 enzymes in the metabolism of drugs and the activation of procarcinogens in human tissues, but very frequently these enzymes have substantial overlapping selectivity. We evaluated a chemically diverse set of nine previously identified CYP2A6 inhibitors to determine which are able to discriminate between human CYP2A enzymes CYP2A6 and the 94%-identical CYP2A13 enzyme. Inhibitor binding to recombinant purified enzyme was evaluated, and affinities were determined.

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The High-Level Expression in Escherichia coli of the Membrane-Bound Form of Human and Rat Cytochrome b5 and Studies on Their Mechanism of Function

Cited by 95 publications

References 0 publications

NAD(P)H:Quinone Oxidoreductase-1-Dependent and -Independent Cytotoxicity of Potent Quinone Cdc25 Phosphatase Inhibitors

NAD(P)H:Quinone Oxidoreductase-1-Dependent and -Independent Cytotoxicity of Potent Quinone Cdc25 Phosphatase Inhibitors

Stereo- And Regioselectivity Account for the Diversity of Dehydroepiandrosterone (Dhea) Metabolites Produced by Liver Microsomal Cytochromes P450

Evaluation of Inhibition Selectivity for Human Cytochrome P450 2A Enzymes

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