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M J Coon

Publications and source records attributed to M J Coon.

211 records · Page 12Linked to original sources
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The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature.

We provide here a list of 221 P450 genes and 12 putative pseudogenes that have been characterized as of December 14, 1992. These genes have been described in 31 eukaryotes (including 11 mammalian and 3 plant species) and 11 prokaryotes. Of 36 gene families so far described, 12 families exist in all mammals examined to date. These 12 families comprise 22 mammalian subfamilies, of which 17 and 15 have been mapped in the human and mouse genome, respectively. To date, each subfamily appears to represent a cluster of tightly linked genes. This revision supersedes the previous updates [Nebert et al., DNA 6, 1-11, 1987; Nebert et al., DNA 8, 1-13, 1989; Nebert et al., DNA Cell Biol. 10, 1-14 (1991)] in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene and cDNA, we recommend that the italicized root symbol "CYP" for human ("Cyp" for mouse), representing "cytochrome P450," be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen should precede the final number in mouse genes. "P" ("p" in mouse) after the gene number denotes a pseudogene. If a gene is the sole member of a family, the subfamily letter and gene number need not be included. We suggest that the human nomenclature system be used for all species other than mouse. The mRNA and enzyme in all species (including mouse) should include all capital letters, without italics or hyphens. This nomenclature system is identical to that proposed in our 1991 update. Also included in this update is a listing of available data base accession numbers for P450 DNA and protein sequences. We also discuss the likelihood that this ancient gene superfamily has existed for more than 3.5 billion years, and that the rate of P450 gene evolution appears to be quite nonlinear. Finally, we describe P450 genes that have been detected by expressed sequence tags (ESTs), as well as the relationship between the P450 and the nitric oxide synthase gene superfamilies, as a likely example of convergent evolution.

Alleles↗
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Substituted pyridines: nonsteroidal inhibitors of human placental aromatase cytochrome P-450.

The binding to human placental aromatase cytochrome P-450 and resulting extent of inhibition was examined for pyridine, pyridines substituted at the 2-, 3-, or 4-positions with phenyl or benzoyl groups, and the nonpyridinic structural analogs biphenyl and benzophenone. Spectral binding studies with partially purified aromatase indicated that pyridine, 3- and 4-phenylpyridines, and 3- and 4-benzoylpyridines interact at the active site via ligation of the pyridinic nitrogen to heme iron, as judged by the type II difference spectra. The apparent dissociation constants for these compounds are 2.1, 0.11, 0.09, 2.6, and 0.27 mM, respectively. Biphenyl, benzophenone, and 2-benzoylpyridine show exclusively a hydrophobic interaction in the presence of the substrate, androst-4-ene-3,17-dione, to give reverse type I difference spectra, with apparent spectral dissociation constants of 0.14, 0.42, and 1.8 mM, respectively. 2-Phenylpyridine shows a unique spectrum, previously not observed with cytochrome P-450, that has components of both type I and type II spectra. As measured by tritium ion release from [1 beta-3H]androst-4-ene-3,17-dione, competitive inhibition of human microsomal aromatase activity was observed with pyridine, 2-, 3-, and 4-phenylpyridines, biphenyl, and benzophenone, with Ki values of 320, 62, 1.48, 0.36, 750, and 130 microM, respectively. In contrast, 2-, 3-, and 4-benzoylpyridines exhibited complex kinetic behavior from which inhibition constants could not be obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Aromatase Inhibitors↗
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Induction of P-450 cytochromes 2E2, 1A1, and 1A2 by imidazole in neonatal rabbits.

Cytochrome P-450 2E1 is induced in adult rabbits by treatment with alcohol, imidazole, and a variety of other agents, as shown earlier in this laboratory, but it is not known whether the highly homologous P-450 2E2 is similarly induced. In this study, the effects of imidazole on 2E2 expression were examined in neonatal rabbits, in which 2E1 is not detectable. Treatment of the animals with imidazole on days 8 through 11 after birth caused a 3-fold increase in the content of total P-450 in liver microsomes. In contrast, the microsomal content of cytochrome b5 and NADPH-P450 reductase was not changed. Immunoblot analysis revealed a significant increase in the level of P-450 2E2 (3-fold) as well as 1A1 (> 10-fold) and 1A2 (> 2-fold) in hepatic microsomes from imidazole-treated neonatal rabbits. The rates of microsomal N-demethylation of N-nitrosodimethylamine and O-deethylation of 7-ethoxyresorufin were similarly increased from 1.3 and 0.03 nmol/min/mg protein, respectively, to 5.6 and 0.24 nmol/min/mg protein, respectively, by imidazole treatment. Blot analysis indicated that the levels of 2E2, 1A1, and 1A2 mRNAs are not increased by imidazole treatment and that 2E1 mRNA is not detectable in either untreated or imidazole-treated neonates. The induction of P-450 2E2 was confirmed by NH2-terminal amino acid sequence analysis of immunopurified 2E protein from hepatic microsomes of imidazole-treated neonatal rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
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Induction of cytochrome P-450 isozyme 3a (P-450IIE1) in rabbit olfactory mucosa by ethanol and acetone.

Cytochrome P-450 isozyme 3a, the alcohol-inducible form of cytochrome P-450 (P-450IIE1), was previously identified in rabbit nasal microsomes with the use of immunochemical techniques; the occurrence of this cytochrome in the nasal mucosa was subsequently confirmed through RNA hybridization experiments. However, in contrast to the well established inducibility of isozyme 3a in liver and kidney by alcohol treatment of the animals, no induction was observed in the nasal tissue with the use of a polyclonal anti-3a antibody for immunochemical quantitation. Recently, two new P-450 isozymes, designated NMa and NMb, were identified in rabbit nasal microsomes, and were found to have overlapping substrate specificity with isozyme 3a. Moreover, the two new cytochromes cross-react with the polyclonal anti-3a antibody that was used in the earlier study for quantitation of nasal isozyme 3a. These recent findings invalidate our previous conclusion that isozyme 3a is not induced by ethanol treatment of rabbits. In the present study, immunoblot quantitation of isozyme 3a was performed with a monoclonal anti-3a antibody that does not recognize either NMa or NMb, and the nasal microsomal metabolism of butanol was examined at various substrate concentrations. We have found that the level of isozyme 3a protein in nasal mucosa is elevated about 2-fold after treatment of the animals with ethanol and about 6-fold after treatment with acetone. Furthermore, corresponding increases in the rate of microsomal butanol oxidation were observed at low substrate concentrations. Thus, we conclude that P-450 isozyme 3a is, in fact, inducible in the nasal tissues by ethanol or acetone treatment of rabbits.

Acetone↗
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Selective mechanism-based inactivation of the major phenobarbital-inducible P-450 cytochrome from rabbit liver by phencyclidine and its oxidation product, the iminium compound.

Phencyclidine, 1-(1-phenylcyclohexyl)piperidine, was found in this study to be a mechanism-based inactivating agent for cytochrome P-450 form 2, the major phenobarbital-inducible cytochrome of rabbit liver microsomes. This process is highly selective for P-450 form 2, both in microsomes and in the reconstituted enzyme system, in that forms 3a, 3b, 4, and 6 are not affected. However, phencyclidine iminium ion, an oxidative metabolite, inactivates both P-450 form 2 and form 3b in a metabolism-dependent manner. Both phencyclidine and its iminium ion give biphasic kinetics of inactivation with similar rate constants, which supports the hypothesis that the iminium ion is an intermediate in the inactivation of P-450 form 2 by the parent compound. The absorption of the oxidized cytochrome and of the ferrous-carbonyl complex in the visible spectrum are both decreased upon inactivation by phencyclidine, indicating modification of the heme moiety. Several modified hemes produced by the action of phencyclidine were isolated by HPLC.

Animals↗
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Evaluation of the role of free hydroxyl radicals in the cytochrome P-450-catalyzed oxidation of benzene and cyclohexanol.

The possible role of free hydroxyl radicals in the oxidation of cyclohexanol to cyclohexanone and of benzene to phenol was examined in a reconstituted system containing rabbit phenobarbital-inducible P-450LM2. From steady state kinetic studies, a KM for cyclohexanol of 8.7 mM and a Vmax of 5.7 nmol of cyclohexanone formed/min/nmol of P-450 were determined. Similarly, a KM for benzene of 105 mM and a Vmax of 22 nmol of phenol formed/min/nmol of P-450 were obtained. With intact microsomes from phenobarbital-treated rabbits, a KM for benzene of 18 mM and a Vmax of 1.7 nmol of phenol formed/min/nmol of P-450 were determined. With the use of substrate concentrations in the range of the respective KM values, superoxide dismutase, desferrioxamine, and dimethyl sulfoxide were found to have no significant effect on the P-450-catalyzed reactions. When the oxidation of benzene or cyclohexanol was examined in a model hydroxyl radical-generating system containing xanthine, xanthine oxidase, and Fe-EDTA, no dependence of the rate of oxidation on the substrate concentrations used was observed. Since the rate of hydroxyl radical generation by the model system was adjusted to be greater than the rate of product formation in the P-450 system, the lack of dependence on substrate concentration suggests that free hydroxyl radicals are not involved in the P-450-catalyzed reactions studied. Taken together, these findings indicate that the free hydroxyl radical-mediated pathway observed by other investigators does not contribute significantly to product formation when these substrates are present at concentrations within the range of their respective KM values.

Animals↗
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Effects of conditions for reconstitution with cytochrome b5 on the formation of products in cytochrome P-450-catalyzed reactions.

Evidence is presented that the method of reconstitution of the cytochrome P-450-containing liver microsomal enzyme system with cytochrome b5 (b5), including the order of addition of the components, the concentration of the b5, and the length of incubation prior to initiation of the reaction by NADPH, governs the steady state catalytic activity obtained. For example, the addition to cytochrome P-450 isozyme 2, NADPH-cytochrome P-450 reductase, and phosphatidylcholine of concentrated b5 (0.4 microM) results in extensive inhibition of benzphetamine demethylation and NADPH oxidation, whereas the addition of dilute b5 (0.02 microM) to the other components results in extensive stimulation of the demethylation reaction. The inhibition is partly relieved by prolonged incubation. The effects of pH and buffer concentration were determined, and the optimal molar ratio of b5 to cytochrome P-450 isozyme 2 was shown to be about 2.0 for stimulation of benzphetamine demethylation, dimethylaniline demethylation, and cyclohexanol oxidation to cyclohexanone. Cytochrome P-450 isozyme 4-catalyzed aminopyrine demethylation and aniline p-hydroxylation are not stimulated by b5, as predicted from a model based on stopped flow kinetic measurements [Pompon and Coon: J. Biol. Chem. 259, 15377 (1984)]. End-point stoichiometry measurements were carried out with cytochrome P-450 isozyme 2 in the absence of b5 or in the presence of b5 under optimal conditions. The results indicate that when b5 is reconstituted with the cytochrome P-450 isozyme 2 enzyme system under optimal conditions, substrate monooxygenation is enhanced, NADPH oxidation is unaffected, and hydrogen peroxide formation is decreased.

Aminopyrine↗
PubMedDate not supplied

Cytochrome P-450 isozymes 2 and 5 in rabbit lung and liver. Comparisons of structure and inducibility.

Rabbit cytochrome P-450 isozymes 2 and 5 were purified from pulmonary and hepatic microsomal preparations. Purification of isozyme 5 was monitored by immunochemical methods so that contamination by isozymes 2, 4, and 6 could be avoided. Partial proteolysis of hepatic and pulmonary isozyme 5 showed minor differences in peptide formation when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and visualized by the silver staining method. In contrast, identical patterns were observed when the peptides were transferred to nitrocellulose paper and visualized immunochemically. The differences observed between the results obtained with the two methods was apparently caused by differences in small amounts of contaminants present in both preparations. HPLC profiles of peptides formed by treatment of pulmonary and hepatic isozyme 5 with trypsin appeared to be the same. In addition, it was found that the pulmonary and hepatic isozymes had identical sequences for the first 20 NH2-terminal amino acids. Three distinct fractions of hepatic cytochrome P-450 isozyme 2 were obtained when chromatography on DEAE-cellulose was used as the final step in the purification procedure. In contrast, only a single fraction of purified pulmonary isozyme 2 was isolated by the same method. Analysis of the pulmonary and three hepatic preparations of isozyme 2 by partial proteolysis and visualization of peptides by silver staining or immunoblotting showed no differences. Analysis of tryptic digests by HPLC also produced the same results for each of the four preparations. The first 24 NH2-terminal amino acids were identical for all four preparations of isozyme 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
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Cytochrome P-450-dependent formation of ethylene from N-nitrosoethylamines.

Ethylene was identified as a metabolite of N-nitrosodiethylamine (NDEA) as well as of N-nitrosomethylethylamine and N-nitrosoethylbutylamine. The formation of ethylene from these carcinogenic compounds is cytochrome P-450- and NADPH-dependent. With NDEA, the rate of the reaction in rabbit liver microsomes is increased by treatment of the animals with phenobarbital or ethanol, which are known to induce the synthesis of P-450 form 2 and P-450 form 3a (P-450ALC), respectively, and the activity is inhibited by anti-P-450 form 2 or form 3a IgG. The rate of ethylene formation is about 10-fold higher in nasal microsomes than in hepatic microsomes. In a reconstituted system, P-450 form 3a is the most active in ethylene formation from NDEA, followed by forms 2 and 4. It has been proposed by others that the alpha-hydroxylation of N-nitrosodialkylamines leads to the formation of an aldehyde and an alkyl diazonium ion, which, with the loss of N2, yields an alkyl carbonium ion capable of producing the corresponding alcohol or binding macromolecules through electrophilic reactions. In the case of the N-nitrosoalkylethylamines we have studied, the ethyl carbonium ion produced could, in addition, generate ethylene by proton elimination.

Animals↗
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Effects of cytochrome b5 on cytochrome P-450-catalyzed reactions. Studies with manganese-substituted cytochrome b5.

The effects of cytochrome b5 with manganese-protoporphyrin IX substituted for heme were compared with those of native cytochrome b5 and the apoenzyme on the oxygenation of substrates in the reconstituted system containing liver microsomal cytochrome P-450, NADPH-cytochrome P-450 reductase, and phosphatidylcholine. Mn-b5, unlike b5, remains essentially fully oxidized in the presence of NADPH and NADPH-cytochrome P-450 reductase under aerobic conditions. The effects of various concentrations of b5 and its derivatives were determined at constant P-450 and reductase concentrations. Cytochrome b5 inhibits benzphetamine demethylation by isozyme 2, the effect increasing up to the highest concentrations tested, and stimulates 7-ethoxycoumarin deethylation by isozyme 2 and acetanilide p-hydroxylation by isozyme 4, the optimal b5:P-450 molar ratio being about 2. In contrast, Mn-b5 inhibits all three reactions and apo-b5 is either inactive or slightly inhibitory. The activities of the three substrates as well as testosterone were determined with P-450 isozymes 2, 3b, 3c, and 4 in the reconstituted system with no additions or with b5 or Mn-b5 present. Cytochrome b5 is stimulatory, inhibitory, or without any effect, the result depending on both the substrate and P-450 isozyme present, whereas Mn-b5 is inhibitory in most instances. Both b5 and its manganese derivative alter the rates of testosterone 6 beta- or 16 alpha-hydroxylation by most of the P-450 cytochromes. The activities are influenced by the molar ratio of reductase to P-450. The Km values of benzphetamine, ethoxycoumarin, and acetanilide are, with one exception, significantly decreased in the presence of b5 or Mn-b5. We conclude that some of the effects of b5 on the oxygenase system are not accounted for by its role as an electron donor to cytochrome P-450.

Animals↗