Search PubMed⌕ Search

Biomedical subjects

M J Coon

Publications and source records attributed to M J Coon.

At least 73 records · Page 4Linked to original sources
PubMed1990

Immunochemical characterization of multiple forms of cytochrome P-450 in rabbit nasal microsomes and evidence for tissue-specific expression of P-450s NMa and NMb.

Two unique forms of cytochrome P-450 (P-450), designated NMa and NMb, were recently isolated in this laboratory from nasal microsomes of rabbits. In the present study, polyclonal antibodies to the purified nasal cytochromes were prepared. Immunochemical analysis with specific rabbit anti-NMa and sheep anti-NMb antibodies indicated that P-450 isozymes identical to or having a high structural homology with NMa are present in both olfactory and respiratory mucosa, as well as in liver, but NMb was detected only in the olfactory mucosa. Neither form was detected in other tissues examined, including brain, esophageal mucosa, heart, intestinal mucosa, kidney, and lung. The specific occurrence of NMb in the olfactory mucosa was further substantiated by the detection and specific inhibition by anti-NMb of the formation of unique NMb-dependent metabolites of testosterone in olfactory microsomes but not in microsomes from liver or respiratory mucosa. Similar experiments with antibodies to previously purified rabbit hepatic P-450 isozymes indicated that not all of the hepatic cytochromes are expressed in the nasal tissues. Thus, P-450 isozymes structurally homologous to hepatic forms 2, 3a, and 4, but not 3b and 6, were found in the olfactory mucosa. On the other hand, only form 2 was detected in the respiratory mucosa. Immunoquantitation experiments revealed that NMa and NMb are the major P-450 forms in olfactory microsomes, whereas NMa and P-450 form 2 (or its homolog) constitute the major portion of the respiratory nasal microsomal P-450. The level of NMa in the liver is relatively low, accounting for less than 3% of total microsomal P-450 in this tissue. In addition, evidence is provided that NMa is the major catalyst in the dealkylation of two nasal carcinogens, hexamethylphosphoramide and phenacetin, in both olfactory and respiratory nasal microsomes.

Animals↗
PubMed1989

On the mechanism of action of cytochrome P-450. Spectral intermediates in the reaction with iodosobenzene and its derivatives.

Cytochrome P-450 is known to catalyze the following oxygen transfer reaction: RH + PhIO----ROH + PhI where RH represents a variety of hydroxylatable substrates and PhIO a variety of iodosobenzene derivatives that serve as oxygen donors, and neither molecular oxygen nor an external electron donor is required. To determine whether the cytochrome functions in such reactions by a peroxidase-type mechanism, the kinetics of its interaction with a variety of substituted iodosobenzenes and iodobenzene diacetates have been determined by stopped flow spectrophotometry. The reaction of phenobarbital-induced rabbit liver microsomal cytochrome P-450 form 2 with iodosobenzenes or iodobenzene diacetates leads to the reversible formation of three spectral intermediates, termed E, F, and G. Complex E is characterized by a type I difference spectrum, representing the iodosobenzene-dependent partial shift of the low spin hexacoordinate form of the ferric enzyme to the high spin pentacoordinate form, F represents a transient intermediate whose spectrum cannot be determined for kinetic reasons, and G represents a blue-shifted intermediate with an absorption maximum at about 393 nm in the absolute spectrum. The striking and principal feature of these observations is that the spectrum of Complex G does not vary with structural differences in the iodosobenzene derivatives, in contrast to the transient species observed in previous studies in this laboratory in the reaction between cytochrome P-450 form 2 and aromatic peroxy compounds. Complex G exhibits the spectral properties one might anticipate for an iron-oxo intermediate containing only one oxygen atom derived from the starting iodosobenzene.

Animals↗
PubMed1989

Studies on covalent binding of (-)trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to cytochromes P-450 LM2 and LM4 and NADPH-cytochrome P-450 reductase.

1. Metabolism of 14C-labelled benzo[a]pyrene (-)trans-7,8-dihydrodiol to protein- and DNA-binding products in a reconstituted enzyme system proceeds 5 to 10 times faster with rabbit cytochrome P-450 LM4 than with LM2. 2. Either cytochrome converts the substrate to ethyl acetate- and water-soluble metabolites, identified by h.p.l.c. Water-soluble metabolites comprise 78% of the total products with cytochrome P-450 LM2, but only 50% of those formed by LM4. The relative proportion of the two types of metabolites is differentially affected by certain modifiers such as 7,8-benzoflavone. 3. Half of the radioactivity in the aqueous phase of reaction mixtures containing cytochrome P-450 LM4 represents (-)trans-7,8-diol metabolites in complex primarily with NADPH and phosphate. The remaining water-soluble products are bound covalently to proteins in the reconstituted system. 4. Polyacrylamide gel electrophoresis, autoradiography, and measurement of the radioactivity in individual bands indicate that a larger fraction of metabolites is bound to cytochrome P-450 LM4 than to NADPH-cytochrome P-450 reductase, and only marginal binding to cytochrome P-450 LM2 is seen. Metabolite binding to added DNA is likewise substantially greater in magnitude when cytochrome P-450 LM4, as opposed to LM2, catalyses (-)trans-7,8-diol oxygenation. Thus, the degree of metabolite binding to monoxygenase proteins and to DNA correlates well with the catalytic activity of cytochrome P-450 LM4 and LM2 towards (-)trans-7,8-diol. 5. DNA causes a dramatic enhancement in the activity of cytochrome P-450 LM4 with (-)trans-7,8-diol, indicating that the cytochrome and/or the reductase may be functionally impaired by metabolites of this substrate. Such an effect may alter the balance between detoxication and activation of the carcinogenic benzo[a]pyrene.

Animals↗
PubMed1989

Induction and tissue-specific expression of rabbit cytochrome P450IIE1 and IIE2 genes.

Treatment of rabbits with a variety of dissimilar chemicals, including ethanol, acetone, and imidazole, results in elevated levels of hepatic and renal cytochrome P-450 form 3a, also designated P-450ALC or P-450IIE. The P450IIE1 subfamily in rabbits is composed of two genes that encode proteins with 97% sequence identity; the mRNAs from these genes can be distinguished by their differing electrophoretic mobilities. In the present studies, examination of the expression of these genes revealed that P450IIE1 (gene 1) mRNA is present in greatest abundance in the liver, is present in kidney and nasal mucosa at approximately 10% of the level in liver, and is present in lung at approximately 5% of the level in liver. P450IIE2 (gene 2) mRNA is present in liver and lung at approximately 50% of the level of gene 1 mRNA in these tissues but cannot be detected in kidney or nasal mucosa. Neither gene is expressed in testis, ovary, small intestine, or adrenal tissue. Treatment of rabbits with acetone or imidazole results in elevated levels of P-450 3a-immunoreactive protein in liver and kidney without concomitant increases in P450IIE gene mRNAs. Moreover, various lengths of ethanol treatment elevated the level of immunoreactive protein in liver and kidney, with a rapid reduction of gene 1 mRNA and, at 14 days, gene 2 mRNA to approximately 50% of control levels. In contrast to these chemical inducers of 3a, fasting for 48 hr significantly increases gene 1 and 2 mRNA in liver but does not increase the level of immunoreactive protein. These results indicate that the rabbit P450IIE genes are not coordinately expressed or regulated and, as found with the rat ortholog P-450j, chemical inducers of 3a evidently act through changes in the rate of synthesis or degradation of the enzyme, rather than through increased gene transcription.

Animals↗
PubMed1988

Purification and characterization of two unique forms of cytochrome P-450 from rabbit nasal microsomes.

Two forms of cytochrome P-450, designated P-450NMa and P-450NMb, were purified to electrophoretic homogeneity from rabbit nasal microsomes. The purified cytochromes, which contained 14-16 nmol of P-450/mg of protein, exhibited apparent monomeric molecular weights of 49,500 and 51,000, respectively. As indicated by several criteria, including the amino acid composition, absorption spectra, and peptide maps, the two nasal forms of P-450 are distinct from each other. Furthermore, as judged by the NH2-terminal amino acid sequences, they are distinct from all other P-450 cytochromes described to date. In the ferric form, P-450NMa is in the low-spin state, whereas P-450NMb is predominantly in the high-spin state. When reconstituted with NADPH-cytochrome P-450 reductase and phospholipid, P-450NMa is very active in the oxidation of ethanol as well as several nasal procarcinogens, including the N-deethylation of N-nitrosodiethylamine, the O-deethylation of phenacetin, and the N-demethylation of hexamethyl-phosphoramide. P-450NMb also metabolizes these substrates, but at lower rates. Both nasal forms are also active with testosterone, with P-450NMa oxidizing the substrate in the 17-position to give androstenedione and P-450NMb catalyzing hydroxylation in the 15 alpha-, 16 alpha-, and 19-positions. The two cytochromes represent the major portion of the total P-450 in nasal microsomes, but the corresponding forms could not be detected in hepatic microsomes.

Amino Acid Sequence↗
PubMed1988

Organization and differential expression of two highly similar genes in the rabbit alcohol-inducible cytochrome P-450 subfamily.

The exon-intron organization of two rabbit genes that hybridize with cytochrome P-450 3a (P-450ALC) cDNA has been determined by restriction mapping and sequence analysis. Gene 1 encodes cytochrome P-450 3a as judged by the complete identity of its coding nucleotide sequence with P-450 3a cDNA. Gene 2 encodes a previously uncharacterized cytochrome P-450 that is 97% identical in primary structure to P-450 3a, with 16 amino acid differences scattered throughout the protein. Genes 1 and 2, which are 10 and 9 kilobases in length, respectively, are comprised of 9 exons with exon-intron junctions occurring at identical positions along the mRNA sequences. Each gene contains two transcription start sites approximately 27 and 33 nucleotides upstream from the translation initiation codon, as determined by primer extension and S1 nuclease protection experiments. The predicted lengths of gene 1 and 2 transcripts from the first transcription start site to the poly(A) attachment site are 1999 and 1660 nucleotides, respectively. This difference in size is primarily the result of a 338-base pair deletion in the 3' nontranslated portion of the gene 2 transcript relative to that of gene 1. The two genes show considerable similarity in their 5' flanking regions, including a "TATAA" transcriptional promoter element at position -28. However, a 32-base pair element that is repeated in gene 1 is present only as a single inexact copy in gene 2. By use of synthetic oligonucleotides as hybridization probes, gene 2 transcripts were shown to be present in poly(A)+ RNA from untreated rabbit liver at approximately 50% of P-450 3a mRNA levels. In kidney, however, no gene 2 mRNA was detected although 3a mRNA was present at approximately 10% of the level in liver.

Amino Acid Sequence↗
PubMed1988

Isolation and partial characterization of the gene for cytochrome P-450 3a (P-450ALC) and a second closely related gene.

Two genes that hybridize to the cDNA for alcohol-inducible cytochrome P-450 form 3a (P-450ALC) have been isolated from a rabbit genomic library and characterized by restriction mapping, hybridization, and partial sequence analysis. The genes show extensive sequence similarity as judged by hybridization at high stringency to the coding region of P-450 3a cDNA. However, only gene 1 hybridizes under these conditions to the 3' nontranslated segment of P-450 3a cDNA. The hybridizing fragments derived from both cloned genes were found to be present in the genome of all rabbits examined by Southern blot analysis, indicating that the genes represent separate loci and are not polymorphic alleles. Partial sequence analysis indicated that gene 1 encodes P-450 3a. Gene 2, if transcribed, would encode a protein with greater than 96% sequence identity with P-450 3a in the NH2-terminal region.

Alleles↗
PubMed1988

Radical intermediates in the catalytic cycles of cytochrome P-450.

Schemes are presented summarizing current knowledge of the mechanism of action of cytochrome P-450 when it functions either as a monooxygenase with molecular oxygen as the oxygen donor or as a peroxygenase with peroxy compounds as the oxygen donor. In the process, a large variety of physiologically occurring and foreign compounds undergo hydroxylation and oxy and peroxy radicals are generated. In addition, cytochrome P-450 catalyzes reductive reactions, including a recently discovered reaction in which organic hydroperoxides are cleaved to yield hydrocarbons and aldehydes or ketones. The reaction is believed to involve homolysis of the oxygen-oxygen bond and generation of an alkoxy radical, with beta-scission of the latter followed by reduction of the secondary radical to the hydrocarbon. Evidence has been obtained that lipid hydroperoxides are physiological substrates for this reductive cleavage reaction catalyzed by cytochrome P-450.

Cytochrome P-450 Enzyme System↗
PubMed1988

Identification of P-450ALC in microsomes from alcohol dehydrogenase-deficient deermice: contribution to ethanol elimination in vivo.

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH-) and -positive (ADH+) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH- than from naive ADH+ animals. Ethanol treatment induced the protein about 3-fold in the ADH+ strain and about 4-fold in the ADH- strain. The antibody to rabbit P-450ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450ALC homolog, increased about 3-fold upon ethanol treatment in the ADH+ strain and about 4-fold in the ADH- strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH+ strain and 1.9-fold and 3.3-fold, respectively, in the ADH- strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH+ and ADH- strains, respectively. After chronic ethanol treatment, P-450ALC could account maximally for 8% of the total ethanol elimination in the ADH+ strain and 22% in the ADH- strain. Further, cytochrome P-450ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

Alcohol Dehydrogenase↗
PubMed1988

Role of cytochrome P-450 in hydrocarbon formation from xenobiotic and lipid hydroperoxides.

Cytochrome P-450 has recently been found to catalyze the reductive cleavage of both xenobiotic hydroperoxides and biologically occurring lipid hydroperoxides. In a reconstituted enzyme system containing phenobarbital-inducible P-450 form 2, NADPH-cytochrome P-450 reductase, and NADPH, the corresponding alcohols are not produced, but instead carbonyl compounds and hydrocarbons. In the following equation for the reaction, X represents any of a variety of alkyl groups and R and R' are either hydrogen or alkyl groups, of which only methyl has been studied so far: XRR'C-OOH + NADPH + H+----XRCO + R'H + H2O + NADP+ The products derived from cumyl, alpha-methylbenzyl, benzyl, and t-butyl hydroperoxides as well as 13-hydroperoxy-9,11-octadecadienoic acid indicate that the reaction involves stepwise one-electron transfer, resulting in homolysis of the peroxide oxygen-oxygen bond. The alkoxy radical thus generated undergoes beta-scission to yield the carbonyl compound and an alkyl radical that is reduced to the alkane. As predicted by this radical mechanism, when the reductive cleavage of cumyl hydroperoxide was carried out in D2O, deuteromethane was formed.

Cytochrome P-450 Enzyme System↗
PubMed1987

Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect.

In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17-3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting [1,7,16-3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of 3H2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16-3H]progesterone and [4-14C]progesterone was employed.

Animals↗
PubMed1987

Alcohol-inducible cytochrome P-450 (P-450ALC).

Of the family of P-450 cytochromes occurring in rabbit liver microsomes, only isozyme 3a (P-450ALC) is induced by alcohol administration and is effective in catalyzing the reaction: ethanol +02+NADPH+H+----acetaldehyde +2H2O+NADP+. As judged by immunochemical quantitation, P-450ALC is also induced in the animals by other diverse agents, including imidazole, trichloroethylene, acetone, pyrazole, and isoniazid. Evidence has been obtained for the occurrence of a protein immunochemically related to P-450ALC in human liver microsomes and of a similar alcohol-inducible protein in the rat and in the normal and alcohol dehydrogenase-deficient deer-mouse. P-450ALC catalyzes the activation of foreign compounds such as acetaminophen, various nitrosamines, and carbon tetrachloride and is therefore believed to play an important role in the enhanced toxicity of these substances accompanying alcohol administration.

Animals↗
PubMed1987

cDNA and derived amino acid sequence of ethanol-inducible rabbit liver cytochrome P-450 isozyme 3a (P-450ALC).

Administration of ethanol to rabbits is known to induce a unique liver microsomal cytochrome P-450, termed isozyme 3a or P-450ALC, which is responsible for the increased oxidation of ethanol and other alcohols and the activation of toxic or carcinogenic compounds such as acetaminophen and N-nitrosodimethylamine. To further characterize this cytochrome P-450 we have identified cDNA clones to isozyme 3a by immunoscreening, DNA hybridization, and hybridization-selection. The cDNA sequence determined from two overlapping clones contains an open reading frame of 1416 nucleotides, and the first 25 amino acids of this reading frame correspond to residues 21-45 of cytochrome P-450 3a. The complete polypeptide, including residues 1 to 20, contains 492 amino acids and has a molecular weight of 56,820. Cytochrome P-450 3a is approximately 55% identical in sequence to P-450 isozymes 1 and 3b and 48% identical to isozyme 2. Hybridization of clone p3a-2 to electrophoretically fractionated rabbit liver poly(A)+ RNA revealed multiple bands, but, with a probe derived from the 3' nontranslated portion of this cDNA, only a 1.9-kilobase band was observed. Treatment of rabbits with imidazole, which increases the content of isozyme 3a, resulted in a transient increase in form 3a mRNA, but this was judged to be insufficient to account for the known 4.5-fold increase in form 3a protein. Genomic DNA analysis indicated that the cytochrome P-450 3a gene does not belong to a large subfamily.

Amino Acid Sequence↗
PubMed1987

Hydrocarbon formation in the reductive cleavage of hydroperoxides by cytochrome P-450.

Evidence is presented that cytochrome P-450 catalyzes the reductive cleavage of hydroperoxides. For example, in a reconstituted system containing rabbit liver microsomal P-450 form 2, NADPH-cytochrome P-450 reductase, and NADPH, cumyl hydroperoxide yields acetophenone and methane, but no cumyl alcohol is formed. The stoichiometry of the reaction and similar results with alpha-methylbenzyl, benzyl, and t-butyl hydroperoxides are in accord with the following general equation, in which X represents an alkyl group and R and R' are either alkyl groups or hydrogen atoms in the starting peroxide: XRR'C-OOH + NADPH + H+----XRCO + R'H + H2O + NADP+. Because 13-hydroperoxy-9,11-octadecadienoic acid yields pentane under these conditions, we propose that the known formation of alkanes and aldehydes in membrane lipid peroxidation involves reductive cleavage by P-450 to give the products predicted by the above equation. The cleavage reaction is thought to involve stepwise one-electron transfer, resulting in homolysis of the peroxide oxygen-oxygen bond and generation of an alkoxy radical, with beta-scission of the latter followed by reduction of the secondary radical to the hydrocarbon. In accordance with this scheme, when the cleavage reaction with cumyl hydroperoxide was done in 2H2O, deuteromethane was formed.

Animals↗
PubMed1987

The P450 gene superfamily: recommended nomenclature.

A nomenclature for the P450 gene superfamily is proposed based on evolution. Recommendations include Roman numerals for distinct gene families, capital letters for subfamilies, and Arabic numerals for individual genes. An updating of this list, which presently includes 65 entries, will be required every 1-2 years. Assignment of orthologous genes is presently uncertain in some cases--between widely diverged species and especially in the P450II family due to the large number of genes. As more is known, it might become necessary to change some gene assignments that are based on our present knowledge.

Alleles↗
PubMed1987

Stereochemistry of the functional group determines the mechanism of aromatase inhibition by 6-bromoandrostenedione.

A selective inhibitor of aromatase (estrogen synthetase) would be a useful pharmacological tool with potential therapeutic application. We have found that 6 alpha-bromoandrostenedione (6 alpha-BrA) is a competitive inhibitor of human placental aromatase with respect to androstenedione, with an apparent Ki of 3.4 nM, while 6 beta-BrA is a mechanism-based irreversible inhibitor with an apparent Ki of 0.8 microM and a kinact of 0.025 min-1. Aromatase activity was measured by tritium release into water from the 1 beta position of [1(-3)H,4(-14)C]androstenedione in reaction mixtures containing NADPH and the aromatase. Time-dependent inhibition was assessed by preincubation of inhibitors with either the 900 X g placental pellet or placental microsomes in the presence of NADPH. Aliquots were taken at intervals, diluted, and assayed for aromatase activity with androstenedione and additional NADPH. The time-dependent inhibition by 6 beta-BrA was dependent on the concentration of this compound and the presence of NADPH, while the addition of excess substrate in the preincubation mixture hindered the inactivation. Both epimers were ineffective in inhibiting rabbit liver microsomal drug-metabolizing activities in a competitive or time-dependent manner. This indicates a high selectivity of 6-BrA inhibition among P-450 cytochromes. These and other 6-substituted androgens may be useful probes into the nature of the active site and mechanism of action of aromatase.

Androstenedione↗