Showing papers on "Monooxygenase published in 1983"

Cytochrome P-450 isozyme 1 from phenobarbital-induced rat liver: purification, characterization, and interactions with metyrapone and cytochrome b5.

Abstract: Cytochrome P-450 isozyme 1 (PB-1) (Mr congruent to 53 000) was purified to apparent homogeneity from phenobarbital (PB)-induced rat liver microsomes, and its spectral, structural, immunochemical, and catalytic properties were determined. PB-1, present in significant amounts in uninduced rat liver microsomes, is induced approximately 2-4-fold by phenobarbital, as compared to the greater than 30-fold induction typical of the major PB isozymes characterized previously. PB-1 was distinguished from the major PB-induced isozymes PB-4 and PB-5 [Waxman, D. J., & Walsh, C. (1982) J. Biol. Chem. 257, 10446-10457] by the absence of a Fe2+-metyrapone P446 complex, by its unique NH2-terminal sequence and distinct peptide maps, by the lack of immuno-cross-reactivity to PB-4, and by its characteristic substrate-specificity profile. Metyrapone effected a saturable enhancement of several PB-1-catalyzed reactions in the reconstituted system [Km(metyrapone) congruent to 200 microM], which varied in magnitude with the substrate, with a maximal stimulation of 5-8-fold in the case of acetanilide 4-hydroxylation. That metyrapone enhanced the corresponding microsomal activities only in cases where the metyrapone-sensitive PB-4 did not catalyze the same reaction at significant rates suggested that PB-1 is probably responsible for the substrate-dependent stimulatory effects of metyrapone on microsomal monooxygenations. In contrast to PB-4 and PB-5, PB-1 was characterized by a marked, but not absolute, dependence on cytochrome b5 (b5) for catalytic activity, with 4-7-fold stimulations typically effected by inclusion of stoichiometric b5 in the reconstituted system. That these b5-stimulations were lipid dependent and were abolished with specific proteolytic fragments lacking b5's COOH-terminal membranous segment evidenced the importance of this segment for efficient, b5-mediated electron transfer to P-450 PB-1 in the reconstituted monooxygenase system.

Induction and Characterization of a Microsomal Flavonoid 3′-Hydroxylase from Parsley Cell Cultures

Abstract: A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen-dependent hydroxylation of (S)-naringenin [(S)-5, 7, 4'-trihydroxyflavanone] to eriodictyol (5, 7, 3', 4'-tetrahydroxyflavanone). Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3'-hydroxylase reaction. In contrast prunin (naringenin 7-O-beta-glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4-hydroxylase, did not catalyse hydroxylation of 4-coumaric acid to caffeic acid. 3'-Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P-450-dependent flavonoid 3'-monooxygenase. Pronounced differences in the inhibition of flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase were found with EDTA, potassium cyanide and N-ethylmaleimide. Irradiation of the cell cultures led to increase of flavonoid 3'-hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease. This is similar to light-induction of phenylalanine ammonialyase and cinnamate 4-hydroxylase. In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3'-hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and NADPH--cytochrome reductase. The results prove that flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase are two different microsomal monooxygenases.

N-demethylation of cocaine to norcocaine. Evidence for participation by cytochrome P-450 and FAD-containing monooxygenase.

Abstract: Experiments were conducted to determine which microsomal enzymes are involved in the in vitro hepatic oxidative N-demethylation of cocaine to norcocaine, the first step in the biotransformation of cocaine to its ultimate hepatotoxic metabolite. Cocaine was found to undergo conversion to norcocaine by two alternate pathways, one involving only cytochrome P-450 and the other requiring both cytochrome P-450 and FAD-containing monooxygenase. In the first pathway, cocaine was directly N-demethylated to norcocaine by cytochrome P-450; this reaction was enhanced by phenobarbital induction and was inhibited by both n-octylamine and metyrapone. The second route was found to be a two-step reaction involving cocaine N-oxide as an intermediate. In this pathway, cocaine is first oxidized to cocaine N-oxide by FAD-containing monooxygenase, followed by a cytochrome P-450-catalyzed N-demethylation to norcocaine. This latter step was enhanced by phenobarbital treatment and inhibited by n-octylamine. Cocaine N-oxide also exhibited a Type I binding spectrum with mouse hepatic microsomes. In addition, a model system consisting of ferrous sulfate was found to catalyze the N-demethylation of cocaine N-oxide. On the basis of these experiments, it is concluded that cytochrome P-450 and FAD-containing monooxygenase participate in the initial oxidation of cocaine to norcocaine. We also propose a mechanism to account for the conversion of cocaine N-oxide to norcocaine.

Camphor revisited: involvement of a unique monooxygenase in metabolism of 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid by Pseudomonas putida.

Abstract: Previously, Pseudomonas putida was shown to degrade (+)-camphor, and cleavage of the first ring of the bicyclic structure involved two monooxygenases (a hydroxylase and a ring oxygen-inserting enzyme), a dehydrogenase, and spontaneous cleavage of an unstable oxygenation product (lactone). Cleavage of the second ring was not demonstrated but was assumed also to occur by ring oxygen insertion, since the predicted oxygenation product was extracted from whole-cell incubation systems. Our investigation established that metabolism of the first ring cleavage intermediate, 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid, occurred through the sequential action of two inducible enzymes, a coenzyme A ester synthetase and an oxygenase. The oxygenase was purified to homogeneity and had a molecular weight of 106,000. This enzyme carried a single molecule of flavin adenine dinucleotide and consisted of two identical subunits. Iron was not present at a significant level. The oxygenase was specific for NADPH as the electron donor and absolutely specific for the coenzyme A ester of 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid as the substrate. The reaction stoichiometry was compatible with this enzyme being a monooxygenase, and a mass spectral analysis of the methyl ester of the product confirmed the insertion of a single oxygen atom. The enzyme appeared to be analogous to, although distinct from. 2,5-diketocamphane 1,2-monooxygenase in catalyzing a "biological Baeyer-Villiger" reaction with the formation of a lactone. Structural analogy suggested that this lactone, like the first, was also unstable and susceptible to spontaneous ring opening, although this was not experimentally established.

Xenobiotic metabolism in Clara cells and alveolar type II cells isolated from lungs of rats treated with beta-naphthoflavone.

Abstract: An investigation of several pathways for xenobiotic metabolism in rat lung cells was carried out using enriched fractions of alveolar type II cells (80% purity) and Clara cells (50% purity) which had been prepared from either untreated (control) rats or animals which had been treated with beta-naphthoflavone. Monooxygenase activities (7-ethoxycoumarin deethylase; aryl hydrocarbon [benzo(a)pyrene] hydroxylase) and activities of conjugating enzymes (glutathione transferase; glucuronosyl transferase) were found to be much higher in fractions enriched in Clara cells than in either the crude cell digest or in fractions enriched in type II cells. This was also found to be true for epoxide hydrolase activity. beta-Naphthoflavone treatment of animals was found to increase the monooxygenase and glucuronosyl transferase activities in all cell fractions, but no effect was seen on either glutathione transferase or epoxide hydrolase activity, each of which was extremely low in type II cells.

Aerobic and Anaerobic Catabolism of Vanillic Acid and Some Other Methoxy-Aromatic Compounds by Pseudomonas sp. Strain PN-1.

Abstract: Vanillic acid (4-hydroxy-3-methoxybenzoic acid) supported the anaerobic (nitrate respiration) but not the aerobic growth of Pseudomonas sp. strain PN-1. Cells grown anaerobically on vanillate oxidized vanillate, p-hydroxybenzoate, and protocatechuic acid (3,4-dihydroxybenzoic acid) with O(2) or nitrate. Veratric acid (3,4-dimethoxybenzoic acid) but not isovanillic acid (3-hydroxy-4-methoxybenzoic acid) induced cells for the oxic and anoxic utilization of vanillate, and protocatechuate was detected as an intermediate of vanillate breakdown under either condition. Aerobic catabolism of protocatechuate proceeded via 4,5-meta cleavage, whereas anaerobically it was probably dehydroxylated to benzoic acid. Formaldehyde was identified as a product of aerobic demethylation, indicating a monooxygenase mechanism, but was not detected during anaerobic demethylation. The aerobic and anaerobic systems had similar but not identical substrate specificities. Both utilized m-anisic acid (3-methoxybenzoic acid) and veratrate but not o- or p-anisate and isovanillate. Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid), 3-O-methylgallic acid (3-methoxy-4,5-dihydroxybenzoic acid), and 3,5-dimethoxybenzoic acid were attacked under either condition, and formaldehyde was liberated from these substrates in the presence of O(2). The anaerobic demethylating system but not the aerobic enzyme was also active upon guaiacol (2-methoxyphenol), ferulic acid (3-[4-hydroxy-3-methoxyphenyl]-2-propenoic acid), 3,4,5-trimethoxycinnamic acid (3-[3,4,5-trimethoxyphenyl]-2-propenoic acid), and 3,4,5-trimethoxybenzoic acid. The broad specificity of the anaerobic demethylation system suggests that it probably is significant in the degradation of lignoaromatic molecules in anaerobic environments.

Phosphorylation of cytochrome-P-450-dependent monooxygenase components.

Abstract: Most chemical carcinogens require activation by polysubstrate monooxygenase. The phosphorylation of essential components of this cytochrome P-450 monooxygenase system, isolated from rabbit liver microsomes, cytochrome P-450 (LM2) and cytochrome reductase, was tested using two different protein kinases. One of the kinases, a cyclic AMP-independent phosvitin kinase (kinase P), was inactive in all systems tested. However, the catalytic subunit of a cyclic AMP-dependent protein kinase (kinase C) catalyzed phosphoryl group transfer to both proteins, but to different extents. Cytochrome P-450 was phosphorylated when added as sole component and also when in the presence of P-450 reductase and phosphatidylcholine. In contrast, the weak phosphorylation of P-450 reductase was reduced considerably in a complete reconstituted system containing P-450 and phosphatidylcholine. The inclusion of kinase P did not alter these results which excludes the possibility that these kinases participate in a sequential phosphorylation mechanism. The monooxygenase constituents themselves were without kinase activity. When hepatic microsomes were isolated in presence of the phosphatase inhibitor sodium fluoride no significant change in monooxygenase (7-ethoxycoumarin O-deethylation) activity was observed, whilst after preincubation with either acid or alkaline phosphatase a significant reduction in monooxygenase activity was measured. Thus, cytochrome P-450 (LM2) is phosphorylatable by protein kinase C and the catalytic activity of polysubstrate monooxygenase decreases after preincubation of microsomes with phosphatases.

Specificity of rat liver cytochrome P-450 isozymes in the mutagenic activation of benzo[a]pyrene, aromatic amines and aflatoxin B1.

Abstract: The ability of three purified forms of rat liver cytochrome P-450 to metabolically activate benzo[a]pyrene, trans-benzo-[a]pyrene-7,8-dihydrodiol, 2-aminofluorene, aflatoxin B1, dimethylnitrosamine, and a pyrolysis product of tryptophan(3-amino-1-methyl-5H-pyrido(4,3-b)indole) (Trp-P-2) to mutagenic products was examined using Salmonella typhimurium strains TA98 and G46 in a reconstituted monooxygenase system. The isozymes examined were cytochrome P-450-PB (the major phenobarbital inducible form), and the two major 3-MC inducible forms (cytochromes P-448(52) and P-448(55)). Cytochromes P-448(52) and P-448(55) preferentially metabolize 2-aminofluorene and Trp-P-2 to mutagenic products. However, only cytochrome P-448(55) metabolizes benzo[a]pyrene and its 7,8-dihydrodiol derivative to mutagenic products. Both cytochrome P-448(52) and P-448(55) metabolize aflatoxin B1 to mutagenic products at a much faster rate than cytochrome P-450-PB. Dimethylnitrosamine was not activated by any of the isozymes tested.

Prostaglandin Synthetase and Cytochrome P-450-dependent Metabolism of (±)Benzo(a)pyrene 7,8-Dihydrodiol by Enriched Populations of Rat Clara Cells and Alveolar Type II Cells

Abstract: The metabolism of (±)- trans -7,8-dihydroxy-7,8-dihydrobenzo( a )pyrene (BP-7,8-diol) by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases was studied using enriched fractions of Clara cells and alveolar type II cells from rat lung. Arachidonic acid-fortified fractions enriched in Clara cells and alveolar type II cells metabolized BP-7,8-diol to the 7,10/8,9-tetrol of benzo( a )pyrene and the 7/8,9,10-tetrol of benzo( a )pyrene. These tetrols are formed upon solvolysis of (±)-7β,8α-dihydroxy-9α,10α- epoxy-7,8,9,10-tetrahydrobenzo( a )pyrene (BP diol-epoxide I). Arachidonic acid-dependent metabolism of BP-7,8-diol to BP diol-epoxide I in enriched Clara cells and alveolar type II cells was completely inhibited by indomethacin, a classical inhibitor of prostaglandin synthetase. Enriched Clara cells and alveolar type II cells also metabolized BP-7,8-diol to BP diol-epoxide I in the presence of NADPH. Amounts of BP diol-epoxide I-derived tetrols formed from BP-7,8-diol by the prostaglandin synthetase-dependent and the cytochrome P-450-dependent pathways varied significantly between the two pulmonary cell fractions examined. In fractions enriched in Clara cells, cytochrome P-450-dependent BP-7,8-diol oxidation was higher than was prostaglandin synthetase-dependent BP-7,8-diol oxidation; while in fractions of alveolar type II cells, prostaglandin synthetase-dependent BP-7,8-diol oxidation to BP diol-epoxide I predominated. Pretreatment of rats with β-naphthoflavone resulted in a 2- to 3-fold increase in BP diol-epoxide I formation by prostaglandin synthetase and cytochrome P-450-dependent monooxygenases in both enriched Clara cells and alveolar type II cells. These increases in BP-7,8-diol oxidation to BP diol-epoxide I appear to be due to induction of the two enzymatic pathways in both pulmonary cell types. No qualitative changes in the pattern of BP-7,8-diol metabolism by either enzymatic pathway in enriched Clara cells or alveolar type II cells were observed following β-naphthoflavone treatment. The results suggest that pulmonary prostaglandin synthetase may serve as either an additional or an alternative bioactivating enzyme to the cytochrome P-450-dependent monooxygenases for the formation of reactive chemical carcinogens in the lung.

Immunochemical Detection and Quantitation of Microsomal Cytochrome P-450 and Reduced Nicotinamide Adenine Dinucleotide Phosphate: Cytochrome P-450 Reductase in the Rat Ventral Prostate

Abstract: Treatment with β-naphthoflavone (BNF) was found to induce 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities about 500-fold in the microsomal fraction of the rat ventral prostate but had no effect on aminopyrine N-demethylase or reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activities. Phenobarbital (PB) treatment did not alter any of these enzyme activities. Antibodies raised in rabbits against rat liver cytochrome P-450 reductase (P-450 reductase) and against P-450 BNF-B2 and P-450 PB-B2, the major forms of P-450 isolated from liver microsomes of BNF- and PB-treated rats, respectively, were used to characterize the P-450-dependent monooxygenase system in the rat ventral prostate. Anti-P-450 reductase immunoglobulin G inhibited reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity in prostatic microsomes, and anti-P-450 BNF-B2 but not anti-P-450 PB-B2 immunoglobulin G inhibited the BNF-induced prostatic microsomal 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities. A highly sensitive immunoblotting method was used to quantitate P-450 BNF-B2, P-450 PB-B2, and P-450 reductase in prostatic microsomes. Using this technique, prostatic P-450 reductase with a molecular weight corresponding to that of purified liver P-450 reductase was detected at a level of 0.02 nmol/mg of microsomal protein. In the liver, the same enzyme amounts to 0.2 nmol/mg of microsomal protein. P-450 BNF-B2 was not detected in prostatic microsomes from control or PB-treated rats, whereas a protein band with a molecular weight corresponding to that of purified liver P-450 BNF-B2 was found in prostatic microsomes from BNF-treated rats at a level of 0.05 nmol P-450 per mg microsomal protein. P-450 PB-B2 was not detected in prostatic microsomes from either control, PB-treated, or BNF-treated animals.

Covalent binding of metabolites of 4-ipomeanol to rabbit pulmonary and hepatic microsomal proteins and to the enzymes of the pulmonary cytochrome P-450-dependent monooxygenase system.

Abstract: The covalent binding of metabolites of 4-ipomeanol, a potent lung toxin, to proteins in rabbit pulmonary and hepatic microsomal preparations and in purified monooxygenase systems was investigated. The rate of binding was 12-fold greater in pulmonary preparations than in hepatic preparations. Covalent binding in pulmonary microsomal fractions was inhibited 39 to 49% by antibodies to rabbit pulmonary cytochrome P-450II or P-450I and 90% by antibodies to cytochrome P-450 reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scintillation autoradiography of pulmonary microsomal proteins revealed the presence of heavily labeled bands throughout the molecular weight range (Mr) examined. Two of these bands corresponded in mobility to pulmonary cytochrome P-450I (Mr 52,000) and P-450II (Mr 58,000). In addition, there was a great deal of binding associated with very high molecular weight proteins, probably in the form of cross-linked aggregates which were unable to penetrate the gel matrix. In the absence of cofactor, no binding was observed. Binding was decreased by the addition of the following: antireductase greater than glutathione = NADH (without NADPH) greater than anti-II greater than anti-I. The electrophoretic patterns of the proteins from incubation of [3H]-4-ipomeanol with purified pulmonary P-450-dependent monooxygenase enzymes were also examined. In the complete system, the majority of the binding was associated with high molecular weight species located at the origin and with low molecular weight species that migrated with the tracking dye. In the absence of cofactor, some binding to proteins that corresponded with cytochrome P-450 and P-450 reductase was observed. Protease digestion of incubation mixtures resulted in the migration of all bound material at the dye front.

The expression of different monooxygenases supported by cytochrome P‐450 in neonatal rats and in primary fetal hepatocytes in culture

Abstract: In rat liver, the perinatal development of various monooxygenase activities follows different patterns, depending upon the reaction studied. The ontogeny of the 6 beta-, 7 alpha- and 16 alpha-testosterone hydroxylase activities differs very significantly. Aldrin epoxidase and steroid-metabolizing monooxygenases are expressed in primary fetal rat liver cells in culture after treatment in vitro with dexamethasone. Testosterone is not metabolized by the control cells and is hydroxylated on the 6 beta and 16 alpha positions following the addition of corticoids to the culture medium. The dose and time curves vary according to the hydroxylated position of the steroid. Aldrin epoxidase activity is nearly undetectable in the control cells, but is present and is inducible by phenobarbital following treatment with the corticoid. Phenobarbital induces aldrin epoxidase in the absence of dexamethasone in the culture medium, providing that the cells are pretreated with the corticoid for 48 h. The use of antibodies against the main cytochrome P-450 species purified from adult and phenobarbital-treated rats confirms that a similar cytochrome P-450 can be induced in fetal cells in culture. The perinatal regulation of biological events, such as the expression of the monooxygenases, can be reproduced in fetal rat liver cells in culture; such a model constitutes a unique tool for studying the biochemical mechanisms which control these phenomena.