Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q3SYG4 (C18)
 23,707 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antidepressant drugs that contain alkylaminoalkyl substituents have been associated with serious pharmacokinetic interactions in humans that may be related to the inhibition of cytochrome P450 (P450) enzymes. In this study, the propensity of the tricyclic antidepressant nortriptyline (NOR) to inhibit individual microsomal P450 enzymes in rat liver was investigated to provide a mechanistic explanation for these pharmacokinetic interactions. Enzyme kinetic studies revealed that NOR inhibited steroid 2 alpha-, 6 beta, 7 alpha-, and 16 alpha-hydroxylation in untreated rat liver with Km/Ki ratios of 0.53, 0.59, 0.25, and 0.29, respectively. When the drug was preincubated with microsomes and NADPH before testosterone hydroxylation was conducted, marked increases in the Km/Ki ratios were observed (to 8.8, 3.9, 0.62, and 13, respectively). Thus, enzymic oxidation of NOR enhanced its inhibition capacity against P450 activities. Indeed, the altered Km/Ki ratios indicate 17-, 6.6-, 2.5-, and 47-fold increases in inhibition of the four pathways of testosterone hydroxylation after the biotransformation of NOR to its metabolites. From these experiments it was apparent that testosterone 2 alpha- and 16 alpha-hydroxylations, catalyzed predominantly by P450 2C11, were subject to the most pronounced increase in inhibition. Under these conditions, the apparent content of microsomal P450 was decreased, thus suggesting the formation of a NOR metabolite intermediate (MI) complex with the cytochrome. Further, optical difference spectroscopy of NADPH-supported metabolism of NOR in microsomes and in a reconstituted system incorporating purified P450 2C11 indicated the appearance of an absorbance peak near 454 nm, similar to those produced by triacetyloleandomycin, SKF 525-A, and orphenadrine. Formation of this absorbance peak in microsomes was inhibited by an antibody raised against the male-specific P450 2C11. Because oxidative metabolism of NOR to inhibitory products would not necessarily involve MI complexation, additional experiments were undertaken in which NOR-related free metabolites produced in microsomal incubations were removed on Sep-Pak mini-C18 columns before estimation of testosterone hydroxylation. The principal finding from this experiment was that P450 3A2-dependent steroid 6 beta-hydroxylase activity was inhibited to a much lesser extent after removal of unbound NOR metabolites on Sep-Pak columns (25% inhibition after Sep-Pak extraction, compared with 82% inhibition observed when all NOR metabolites were present during subsequent testosterone hydroxylation); inhibition of P450 2C11-mediated 2 alpha- and 16 alpha-hydroxylation was not noticeably different after Sep-Pak treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Metabolite intermediate complexation of microsomal cytochrome P450 2C11 in male rat liver by nortriptyline. 143 57

The metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), a heterocyclic amine carcinogen detected in cooked meats, was investigated in mice. In 3-methylcholanthrene-induced mice administered 0.1, 1.0 and 10 mg/kg [14C]PhIP (i.p.), urinary and fecal excretion over 24 h accounted for 16% and 42-56% of the dose respectively. Urinary excretion of unchanged parent compound accounted for only 0.5-0.8% of the administered dose. At all doses, the major urinary metabolite was identified as 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate and this metabolite comprised approximately 5% of the dose. Uninduced mice excreted greater than 13% of a 10 mg/kg dose as the sulfate conjugate. Urinary excretion of both 2-amino-1-methyl-6-(4'-hydroxy)-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) and a glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (N-hydroxy-PhIP) was also higher (4-fold) in uninduced versus induced mice. The decreased urinary excretion of P450-derived metabolites via induction contrasted with increased metabolite formation by hepatic microsomal preparations. 4'-Hydroxy-PhIP and N-hydroxy-PhIP were produced in amounts nearly 7- and 3-fold higher respectively by induced versus uninduced microsomal incubations at 50 microM [3H]PhIP. At concentrations less than 10 microM, PhIP was almost exclusively converted by the induced preparations to an unidentified metabolite that was not retained by the C18 column. This metabolite, which also was formed in incubations with either 4'-hydroxy-PhIP or N-hydroxy-PhIP, was produced by microsomes from uninduced animals at a much slower rate. Covalent binding to microsomal protein in incubations with [3H]PhIP was concentration-dependent and 2- to 4-fold higher in induced than uninduced preparations. Covalent binding in liver and kidney of induced mice administered [14C]PhIP was dose dependent. At 10 mg/kg PhIP, adducts were produced at 1.7-fold higher levels in livers of induced versus uninduced mice, but renal binding was higher in uninduced animals. These studies indicate the importance of cytochrome P450 and other xenobiotic enzymes in the metabolism, disposition and activation of PhIP.
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PMID:Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice. 157 15

In contrast to other P450 enzymes purified from rat liver microsomes, purified P450 IIIA1 (P450p) is catalytically inactive when reconstituted with NADPH-cytochrome P450 reductase and the synthetic lipid, dilauroylphosphatidylcholine. However, purified P450 IIIA1 catalyzes the oxidation of testosterone when reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, an extract of microsomal lipid, and detergent (Emulgen 911). The present study demonstrates that the microsomal lipid extract can be replaced with one of several naturally occurring phospholipids, but not with cholesterol, sphingosine, sphingomyelin, ceramide, cerebroside, or cardiolipin. The ratio of the testosterone metabolites formed by purified P450 IIIA1 (i.e., 2 beta-, 6 beta-, and 15 beta-hydroxytestosterone) was influenced by the type of phospholipid added to the reconstitution system. The ability to replace microsomal lipid extract with several different phospholipids suggests that the nature of the polar group (i.e., choline, serine, ethanolamine, or inositol) is not critical for P450 IIIA1 activity, which implies that P450 IIIA1 activity is highly dependent on the fatty acid component of these lipids. To test this possibility, P450 IIIA1 was reconstituted with a series of synthetic phosphatidylcholines. Those phosphatidylcholines containing saturated fatty acids were unable to support testosterone oxidation by purified P450 IIIA1, regardless of the acyl chain length (C6 to C18). In contrast, several unsaturated phosphatidylcholines supported testosterone oxidation by purified P450 IIIA1, and in this regard dioleoylphosphatidylcholine (PC(18:1)2) was as effective as microsomal lipid extract and naturally occurring phosphatidylcholine or phosphatidylserine. These results confirmed that P450 IIIA1 activity is highly dependent on the fatty acid component of phospholipids. A second series of experiments was undertaken to determine whether microsomal P450 IIIA1, like the purified enzyme, is dependent on cytochrome b5. A polyclonal antibody against purified cytochrome b5 was raised in rabbits and was purified by affinity chromatography. Anti-cytochrome b5 caused a approximately 60% inhibition of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation by purified P450 IIIA1 and inhibited these same reactions by approximately 70% when added to liver microsomes from dexamethasone-induced female rats. Overall, these results suggest that testosterone oxidation by microsomal cytochrome P450 IIIA1 requires cytochrome b5 and phospholipid containing unsaturated fatty acids.
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PMID:Cytochrome P450 IIIA1 (P450p) requires cytochrome b5 and phospholipid with unsaturated fatty acids. 165 20

P450 2C2 as well as P450 2E1 [Fukuda, T. et al. (1993) J. Biochem. 113, 7-12] catalyzed the hydroxylation of medium chain fatty acids, although the regioselectivity of substrates of the former contrasted with that of the latter. Whereas P450 2E1 hydroxylated C9-C18 fatty acids at the omega-1 position and to a much lesser extent at the omega and omega-2 positions, P450 2C2 hydroxylated C9-C13 fatty acids at different positions dependent on the chain length of fatty acids. Among the fatty acids used as the substrate, undecanoate was hydroxylated at the omega-1 position almost exclusively by P450 2C2. The proportion of omega-hydroxylated products produced by P450 2C2 was markedly increased with decreasing chain length of fatty acids, while the hydroxylation positions were enlarged to the omega-3 position with tridecanoate. When the conserved Thr at the putative distal helix was replaced with Ser, the substrate regioselectivity of the two P450s was affected in different manners. The mutation of P450 2C2 did not change the hydroxylation positions of C9-C12 fatty acids, but caused a significant decrease in the proportion of the omega-1 hydroxy analog in the total products. In sharp contrast to P450 2C2, the mutated P450 2E1 gave additional products to those with the wild-type P450, and the number of different products increased with increasing chain length of the fatty acids. Thus, the products of palmitate hydroxylation were identified as omega-1, omega-2, omega-3, omega-4, omega-5, omega-6, and omega-7 monohydroxy isomers using gas chromatography-electron impact mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Different mechanisms of regioselection of fatty acid hydroxylation by laurate (omega-1)-hydroxylating P450s, P450 2C2 and P450 2E1. 820 83

Threonine-303 of rabbit P450 2E1, which is putatively located at the distal heme surface, was replaced by serine and valine via site-directed mutagenesis. In the oxidized state, the Ser-mutated P450 exhibited a low- and high-spin mixed-type (low > high) absorption spectrum, whereas the Val-mutated P450, like the wild-type P450, exhibited a nearly high-spin type spectrum. The reduced CO complexes of the Ser- and Val-mutated P450s, as well as that of the wild-type P450, showed a Soret absorption maximum at 452 nm. Both mutated P450s were active in the hydroxylation of C10 to C18 fatty acids at somewhat lower rates than the wild-type P450. The Val-mutated P450 gave the same two products (the major one is probably the omega-1 hydroxy analog) as the wild-type P450, while additional products were formed on incubation with C11 to C17 fatty acids as substrates of the Ser-mutated P450; a total of four products was detected for each of the C12 to C15 fatty acids, and three for each of the C11, C16, and C17 homologues. The metabolites of laurate were determined by GC-MS analysis to be the omega-1, omega-2, omega-3, and omega-4 hydroxy counterparts. The Ser-mutated P450 hydroxylated drug substrates at almost the same rates as the wild-type P450, while the mutation to valine significantly lowered the drug hydroxylase activities.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Replacement of Thr-303 of P450 2E1 with serine modifies the regioselectivity of its fatty acid hydroxylase activity. 845 77

A highly sensitive, simple assay for the determination of 4-nitrocatechol formed during the P450-dependent hydroxylation of p-nitrophenol has been developed. The assay utilizes high-performance liquid chromatography with electrochemical detection. The procedure consists of enzymatic reaction, quenching with trifluoroacetic acid, precipitation of the protein by centrifugation, and direct injection of supernatant aliquots for analysis on a reverse-phase C18 column. The electrochemical detection makes it possible to detect 0.5 pmol of 4-nitrocatechol injected, 30 times less than previously reported for an ultraviolet detector. The detector response was linear with time and with protein content, down to as little as 1.0 microgram of microsomal protein. The usefulness of the method was demonstrated with hepatic microsomal as well as S-9 fractions from both ethanol-treated and control rats, and the dependency of the reaction on cytochrome P450 2E1 activity was proven by immunoinhibition experiments.
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PMID:The determination of cytochrome P450 2E1-dependent p-nitrophenol hydroxylation by high-performance liquid chromatography with electrochemical detection. 878 20

Corticosterone methyloxidase I (CMO I) deficiency is an autosomal recessive disorder of aldosterone biosynthesis. To determine further the molecular genetic basis of CMO I deficiency, a patient of Turkish origin that suffered from CMO I deficiency was studied. Nucleotide sequencing of the PCR-amplified exons from the genomic DNA of this patient revealed a single point mutation CTG (leucine) CCG (proline) at codon 461 in exon 8 of CYP11B2, which is involved in the putative heme binding site of steroid 18-hydroxylase (P450(C18)). The expression study using a cDNA introducing the point mutation revealed that the amino acid substitution totally abolishes the P450(C18)p3 enzyme activities required for conversion of 11-deoxycorticosterone to aldosterone, even though the mutant product was detected in the mitochondrial fraction of the transfected cells. These results suggest that this point mutation causes CMO I deficiency.
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PMID:CMO I deficiency caused by a point mutation in exon 8 of the human CYP11B2 gene encoding steroid 18-hydroxylase (P450C18). 917 80

The A. thaliana EST database was screened using consensus motifs derived from P450 families CYP52 and CYP4 catalyzing the omega-hydroxylation of fatty acids and alkanes in Candida and in mammals. One EST cDNA fragment was detected in this way and the corresponding full-length cDNA was cloned from a cDNA library of A. thaliana. This cDNA coded the first member of a new plant P450 family and was termed CYP86A1. The deduced peptide sequence showed highest homology with P450s from families 4 and 52. To confirm the catalytic function, CYP86A1 was expressed in a yeast overexpressing its own NADPH-P450 reductase. Efficient expression was evidenced by spectrophotometry, SDS-PAGE and catalytic activity. CYP86A1 was found to catalyze the omega-hydroxylation of saturated and unsaturated fatty acids with chain lengths from C12 to C18 but not of hexadecane. Genomic organization analyzed by Southern blot suggested a single gene encoding CYP86A1 in A. thaliana.
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PMID:CYP86A1 from Arabidopsis thaliana encodes a cytochrome P450-dependent fatty acid omega-hydroxylase. 950 Sep 87

Quinazoline type alpha1-adrenoceptor antagonists (range 10-100 microM) inhibited aldosterone release of a cell suspension of porcine adrenocortical cells, potency order: doxazosin > prazosin > trimazosin. Phenoxybenzamine also inhibited the aldosterone release at a concentration of 100 microM. Alpha1-adrenoceptor antagonists from other chemical classes had no measurable effect on the aldosterone output from adrenocortical cells in vitro. Agonists selective for either alpha1- or beta-adrenoceptors did not affect the aldosterone release. The inhibition of the aldosterone release induced by quinazolines was similar with different substrates. The small differences between the drug-induced inhibitions could be ranked as corticosterone = progesterone > pregnenolone = deoxycorticosterone. The doxazosin (10 microM)-induced changes in the release of nine steroids indicated that quinazoline-type alpha1-antagonists interfere with enzymes of the aldosterone biogenesis pathway involved in C18-oxidation and C21beta-hydroxylation, reducing the release of both aldosterone and corticosterone. At higher concentrations (100 microM), the C21beta-hydroxylation in the cortisol biogenesis pathway is also affected, decreasing the output of cortisol and deoxycortisol, but increasing the output of progesterone and OH-progesterone. Simultaneously, the C17-oxidation and side-chain cleavage is also inhibited, decreasing the output of androstenedione. The rank order of phenoxybenzamine (100 microM)-induced inhibition of the aldosterone release with different substrates is pregnenolone > corticosterone = progesterone > deoxycorticosterone. With pregnenolone as substrate, the output of aldosterone, corticosterone, and cortisol was reduced to the same extent. The dehydroepiandrosterone, androstenedione, and progesterone release was enhanced. It seems that phenoxybenzamine is a rather selective inhibitor of the mitochondrial P450(11beta/18) enzymes.
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PMID:Effects of alpha1-antagonists on production and release of aldosterone and other steroid hormones by porcine adrenocortical cells in vitro. 992 6

Chloroacetaldehyde (CA) is a nephrotoxic and neurotoxic metabolite of the anticancer drug ifosfamide (IFA) and is a dose-limiting factor in IFA-based chemotherapy. Plasma levels of CA in IFA-treated cancer patients are often difficult to determine due to the lack of a sufficiently sensitive and specific analytical method. We have developed a simple and sensitive HPLC method with fluorescence detection to measure CA formation catalyzed by liver cytochrome P450 enzymes, either in vivo in IFA-injected rats or in vitro in liver microsomal incubations. This method is based on the formation of the highly fluorescent adduct 1-N(6)-ethenoadenosine from the reaction of CA with adenosine (10 mM) at pH 4.5 upon heating at 80 degrees C for 2 h. The derivatization mixture is directly injected onto a C18 HPLC column and is monitored with a fluorescence detector. Calibration curves are linear (r > 0.999) over a wide range of CA concentrations (5-400 pmol). The limit of detection of CA in plasma using this method is <0.1 microM and only 50 microl of plasma is required for the assay. By coupling this method with a recently described HPLC-fluorescent method to determine acrolein, a cytochrome P450 metabolite of IFA formed during the activation of the drug by 4-hydroxylation, the two major, alternative P450-catalyzed pathways of IFA metabolism can be monitored from the same plasma samples or liver microsomal incubations and the partitioning of drug between these two pathways thereby quantitated. This assay may prove to be useful for studies of IFA metabolism aimed at identifying factors that contribute to individual differences in CA formation and in developing approaches to minimize CA formation while maximizing IFA cytotoxicity.
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PMID:High-performance liquid chromatographic-fluorescent method to determine chloroacetaldehyde, a neurotoxic metabolite of the anticancer drug ifosfamide, in plasma and in liver microsomal incubations. 1045 7


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