Biomaterial Database

A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450. 1985

T Shimada, and J P Shea, and F P Guengerich

A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008862	Microsomes, Liver	Closed vesicles of fragmented endoplasmic reticulum created when liver cells or tissue are disrupted by homogenization. They may be smooth or rough.	Liver Microsomes,Liver Microsome,Microsome, Liver
D010088	Oxidoreductases	The class of all enzymes catalyzing oxidoreduction reactions. The substrate that is oxidized is regarded as a hydrogen donor. The systematic name is based on donor:acceptor oxidoreductase. The recommended name will be dehydrogenase, wherever this is possible; as an alternative, reductase can be used. Oxidase is only used in cases where O2 is the acceptor. (Enzyme Nomenclature, 1992, p9)	Dehydrogenases,Oxidases,Oxidoreductase,Reductases,Dehydrogenase,Oxidase,Reductase
D002384	Catalysis	The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction.	Catalyses
D002855	Chromatography, Thin Layer	Chromatography on thin layers of adsorbents rather than in columns. The adsorbent can be alumina, silica gel, silicates, charcoals, or cellulose. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)	Chromatography, Thin-Layer,Thin Layer Chromatography,Chromatographies, Thin Layer,Chromatographies, Thin-Layer,Thin Layer Chromatographies,Thin-Layer Chromatographies,Thin-Layer Chromatography
D003577	Cytochrome P-450 Enzyme System	A superfamily of hundreds of closely related HEMEPROTEINS found throughout the phylogenetic spectrum, from animals, plants, fungi, to bacteria. They include numerous complex monooxygenases (MIXED FUNCTION OXYGENASES). In animals, these P-450 enzymes serve two major functions: (1) biosynthesis of steroids, fatty acids, and bile acids; (2) metabolism of endogenous and a wide variety of exogenous substrates, such as toxins and drugs (BIOTRANSFORMATION). They are classified, according to their sequence similarities rather than functions, into CYP gene families (>40% homology) and subfamilies (>59% homology). For example, enzymes from the CYP1, CYP2, and CYP3 gene families are responsible for most drug metabolism.	Cytochrome P-450,Cytochrome P-450 Enzyme,Cytochrome P-450-Dependent Monooxygenase,P-450 Enzyme,P450 Enzyme,CYP450 Family,CYP450 Superfamily,Cytochrome P-450 Enzymes,Cytochrome P-450 Families,Cytochrome P-450 Monooxygenase,Cytochrome P-450 Oxygenase,Cytochrome P-450 Superfamily,Cytochrome P450,Cytochrome P450 Superfamily,Cytochrome p450 Families,P-450 Enzymes,P450 Enzymes,Cytochrome P 450,Cytochrome P 450 Dependent Monooxygenase,Cytochrome P 450 Enzyme,Cytochrome P 450 Enzyme System,Cytochrome P 450 Enzymes,Cytochrome P 450 Families,Cytochrome P 450 Monooxygenase,Cytochrome P 450 Oxygenase,Cytochrome P 450 Superfamily,Enzyme, Cytochrome P-450,Enzyme, P-450,Enzyme, P450,Enzymes, Cytochrome P-450,Enzymes, P-450,Enzymes, P450,Monooxygenase, Cytochrome P-450,Monooxygenase, Cytochrome P-450-Dependent,P 450 Enzyme,P 450 Enzymes,P-450 Enzyme, Cytochrome,P-450 Enzymes, Cytochrome,Superfamily, CYP450,Superfamily, Cytochrome P-450,Superfamily, Cytochrome P450
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D006899	Mixed Function Oxygenases	Widely distributed enzymes that carry out oxidation-reduction reactions in which one atom of the oxygen molecule is incorporated into the organic substrate; the other oxygen atom is reduced and combined with hydrogen ions to form water. They are also known as monooxygenases or hydroxylases. These reactions require two substrates as reductants for each of the two oxygen atoms. There are different classes of monooxygenases depending on the type of hydrogen-providing cosubstrate (COENZYMES) required in the mixed-function oxidation.	Hydroxylase,Hydroxylases,Mixed Function Oxidase,Mixed Function Oxygenase,Monooxygenase,Monooxygenases,Mixed Function Oxidases,Function Oxidase, Mixed,Function Oxygenase, Mixed,Oxidase, Mixed Function,Oxidases, Mixed Function,Oxygenase, Mixed Function,Oxygenases, Mixed Function
D001189	Aryl Hydrocarbon Hydroxylases	A large group of cytochrome P-450 (heme-thiolate) monooxygenases that complex with NAD(P)H-FLAVIN OXIDOREDUCTASE in numerous mixed-function oxidations of aromatic compounds. They catalyze hydroxylation of a broad spectrum of substrates and are important in the metabolism of steroids, drugs, and toxins such as PHENOBARBITAL, carcinogens, and insecticides.	Microsomal Monooxygenases,Xenobiotic Monooxygenases,Hydroxylases, Aryl Hydrocarbon,Monooxygenases, Microsomal,Monooxygenases, Xenobiotic
D019388	Cytochrome P-450 CYP1A2	A cytochrome P450 enzyme subtype that has specificity for relatively planar heteroaromatic small molecules, such as CAFFEINE and ACETAMINOPHEN.	CYP1A2,Phenacetin O-Dealkylase,CYP 1A2,Caffeine Demethylase,Cytochrome P-450 LM(4),Cytochrome P-450 LM4,Cytochrome P-450d,Cytochrome P450 1A2,CYP1A2, Cytochrome P-450,Cytochrome P 450 CYP1A2,Cytochrome P 450 LM4,Cytochrome P 450d,Demethylase, Caffeine,O-Dealkylase, Phenacetin,P-450 LM4, Cytochrome,Phenacetin O Dealkylase