Search

The steady state kinetic properties of l-lysine monooxygenase from Pseudomonas fluorescens have been investigated. The enzyme has been shown to exhibit both oxygenase and oxidase activity with different substrates, the oxidase activity being much less efficient. Lysine is an oxygenase-type substrate while ornithine is an oxidase-type substrate (Nakazawa, T., Hori, K. and Hayaishi, O. (1972) J. Biol. Chem. 247, 3439–3444). During steady state analysis, both substrates exhibit sigmoidal saturation curves. Hill plots for lysine and ornithine are biphasic, with the Hill coefficient n = 2.9 at low substrate concentrations and n = 1 at higher concentrations. With lysine as substrate, the reaction does not go to completion, and the per cent completion of the reaction is a function of the initial lysine concentration. e-Aminocaproic acid has been found to act as a nonsubstrate effector by permitting the reaction to reach completion. Furthermore, e-aminocaproate reduces the cooperative effect of lysine, converting the biphasic Hill plot to a monophasic one, with a Hill coefficient of 1 obtaining over most of the concentration range of lysine studied. e-Aminocaproate is also a competitive inhibitor toward lysine with a Ki of 2.6 x 10-4 m. e-N,N-Dimethyl-l-lysine is a poor substrate except in the presence of lysine, in which case the reaction proceeds with both substrates, but stops short of completion, as seen with lysine alone as substrate. These results indicate that lysine itself is an effector, and that at least two sites per flavin are important to catalysis, i.e. one catalytic site and one or more effector sites.

Cinnamic acid 4-hydroxylase in etiolated sorghum seedlings was concentrated in the microsomal or light membrane fraction upon extraction. The three substrates required in the catalysis, cinnamic acid, TPNH, and O2 have Km values in the micromolar range. The same microsomal fraction contained cytochromes P-450 and b5 at 0.10 and 0.55 nmoles · mg of protein-1, respectively. Preparation of a photodissociation action spectrum on carbon monoxide inhibition of the cinnamic acid hydroxylase indicated a requirement for cytochrome P-450 in the catalysis. When challenged with a range of potential inhibitors, the hydroxylase behaved in a manner that is fairly typical of the more extensively studied P-450 monooxygenases of nonplant tissues.

Induction of drug monooxygenase activity by phenobarbital was studied in the mouse isolated small intestine. Incubation for 2 hours in the presence of 5 × 10 −4 M phenobarbital resulted in a 2–3 fold increase of the O -dealkylation activity for 7-ethoxycoumarin based on the protein content of the mucosal cell homogenate. The highest activity was located in the jejunum between 4 and 12 cm distal to the pylorus. The induction started after a lag phase of about 30 min and could be prevented by the simultaneous addition of 1·5 × 10 −5 M puromycin or 5 × 10 −7 M actinomycin D. Puromycin decreased the basal rate of O -dealkylation, but actinomycin D at this concentration did not. Actinomycin D, 2 × 10 −5 M, caused a “superinduction”, i.e. stimulation of the induced O -dealkylation activity 30 per cent greater than that with phenobarbital alone. Incorporation of 14 C-leucine into mucosal proteins was doubled in the presence of phenobarbital but the effect was abolished by inhibitors of protein synthesis. Spectroscopic measurements did not indicate major changes in the content of cytochrome b 5 and cytochrome P-450. However, the activity of NADPH-cytochrome c reductase increased in parallel with the O -dealkylation activity. Hence, the induction can probably be attributed to a more rapid turnover of the monooxygenase cycle.

Tryptophan 5-monooxygenase was purified approximately 1,000-fold from the bovine pineal gland. The purified enzyme catalyzed the hydroxylations of both L-tryptophan and L-phenylalanine at a comparable rate. Evidence was presented suggesting that the hydroxylations of both amino acids were catalyzed by the single enzyme. The apparent Km values for L-tryptophan and for L-phenylalanine were approximately 16 and 32 μM, respectively, when tetrahydrobiopterin was used as a cofactor. The apparent molecular weight of the enzyme was estimated to be approximately 30,000 by gel filtration on columns of Sephadex G-75 and G-100 and by ultracentrifugation in sucrose density gradients. These properties of bovine pineal tryptophan 5-monooxygenase were distinguishable from those of rat liver phenylalanine hydroxylase, another enzyme which had been shown to catalyze the hydroxylations of both L-tryptophan and L-phenylalanine.

Summary Human liver microsomes isolated from operation biopsy samples from patients with cholelithiasis or a malignant disease, were studied with respect to a drug-oxidizing monooxygenase system. The human liver contained about 30-40 mg of microsomal protein per gram of wet weight and about 50% of that was recovered in the microsomal fraction. The mean levels of cytochrome P-450, NADPH-cytochrome c reductase and cytochrome b5 in the livers of cholelithiatic patients were essentially similar to those in the livers of rat and guinea pig and only slightly lower than those in the rabbit liver. The cytochrome P-450-carbon monoxide difference spectrum exhibited a peak at 450 nm and, in microsomes from several smokers, no peak shift to 448 nm was evident. Studies with ethyl isocyanide as a ligand revealed a typical difference spectrum with a peak ratio at pH 7.4 of 455/430 nm of 0.46 and a pH intercept of at about 7.9. There was no correlation between cigarette smoking and a peak ratio. Drug-induced interactions of type I (SKF 525A, bromobenzene), type II (aniline, n-octylamine, KCN) and reverse type I (phenacetin, n-butanol) variety were detected in liver samples studied. Hexobarbital and aminopyrine gave either type I or reverse type I spectral changes. The spectral dissociation constant for aniline was of the same order of magnitude as in rat liver microsomes. These studies seem to indicate that although qualitative differences between hepatic monooxygenase systems in man and laboratory animals may exist, available evidence suggests that many essential differences are only quantitative in nature.

The mobilities of 24 potential metabolites of benzo[ a ]pyrene were examined with high pressure liquid chromatography. Twelve phenols, five quinones, four dihydrodiols, and three oxides were studied. The chromatographic procedure employed allowed the separation and quantitation of benzopyrene metabolites into three major groups consisting of phenols, quinones, and dihydrodiols. Two of the benzopyrene oxides were unstable during chromatography, whereas the third oxide was more stable and chromatographed in the quinone fraction. Treatment of rats with phenobarbital or 3-methylcholanthrene enhanced the metabolism of benzopyrene by liver microsomes and altered the relative amounts of the various metabolites formed. In the absence of epoxide hydrase (EC 4.2.1.63), benzopyrene was metabolized primarily to phenols and quinones but was not appreciably metabolized to dihydrodiols by a solubilized, reconstituted cytochrome P -448 monooxygenase system. Addition of partially purified epoxide hydrase resulted in the formation of benzopyrene dihydrodiols with a concomitant decrease in the formation of phenolic metabolites, indicating that benzopyrene undergoes metabolism via arene oxides that are precursors for dihydrodiols and phenols.

The intraperitoneal administration of aromatic hydrocarbons such as 3-methylcholanthrene or β-naphthoflavone stimulates aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity in C57BL/6N, C57BL/6J, C3H/HeN, BALB/cAnN, CBA/HN, and PL/J inbred mice but not in the DBA/2N, 129/J, RF/J, AKR/N, AKR/J, or AU/SsJ inbred strains. By means of genetic crosses between a "responsive" and a "nonresponsive" strain, the hydroxylase induction appears to segregate as an autosomal dominant trait in the C57BL/6N x RF/J cross, the C57BL/6N x 129/J cross, the C3H/HeN x AKR/N cross, the BALB/cAnN x AKR/N cross, the CBA/HN x AKR/N cross, the BALB/cAnN x DBA/2N cross, the C57BL/6N x AU/SsJ cross, the PL/J x AU/SsJ cross, the C57BL/6J x AKR/J cross, the C57BL/6N x AKR/J cross, and the C57BL/6J x AKR/N cross. The absence of hydroxylase induction appears to segregate in an autosomal dominant fashion in the C57BL/6N x AKR/N cross. The induction process in offspring from the C3H/HeN x DBA/2N cross is inherited additively. In mice from many of the above mentioned crosses, there exists a positive, significant correlation between the induced hepatic hydroxylase activity and (a) new formation of hepatic microsomal cytochrome P1-450; (b) higher inducible hydroxylase activities in lung, kidney, bowel, and skin; and (c) induction of hepatic microsomal 7-ethoxycoumarin O-deethylase, p-nitroanisole-O-demethylase, and 3-methyl-4-methylaminoazobenzene N-demethylase activities. We have found no exceptions to this general observation. The basal hydroxylase activity appears to be inherited additively, within the limits of individual variation. From data involving more than 2200 individual mice of these 12 inbred strains, the induction of the monooxygenase activities and the stimulation of cytochrome P1-450 formation by aromatic hydrocarbons appear to be regulated by at least three alleles at each of two nonlinked genetic loci.

Solubilized cytochrome P-450 monooxygenase and epoxide hydrase activities from rat liver microsomes have been separated by column chromatography. The highly active epoxide hydrase fraction is still contaminated with cytochrome P-450, which has very low monooxygenase activity. The highly purified cytochrome P-450 fraction possesses high monooxygenase activity and is essentially devoid of epoxide hydrase activity. Purification factors for the epoxide hydrase through four purification steps are similar with [3H]styrene oxide, [3H]naphthalene oxide, [3H]cyclohexene oxide, and benzene oxide as substrates. Failure of benzene oxide to inhibit hydration of styrene or naphthalene oxide in the most purified preparations in indicative of the presence of at least two hydrases. These purified cytochrome monooxygenase and hydrase preparations represent valuable tools for the study of the intermediacy of arene oxides in drug metabolism. Thus, with naphthalene, only naphthol is formed with the monooxygenase, while both naphthol and the dihydrodiol are formed in the presence of monooxygenase and hydrase. A convenient radiochemical synthesis of [3H]naphthalene 1,2-oxide and assays for the measurement of the hydration of [3H]naphthalene oxide and benzene oxide, based on differential extractions and high-pressure liquid chromatography, respectively, are described.

The intraperitoneal administration of aromatic hydrocarbons such as 3-methylcholanthrene, β-naphthoflavone, or naphthacene induces several monooxygenase activities and the new formation of a spectrally distinct CO-binding cytochrome in genetically "responsive" inbred mouse strains, but these compounds, even when administered chronically at high doses, fail to induce these changes in genetically "nonresponsive" inbred strains. On the contrary, administration of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) causes induction of a similar magnitude in either "responsive" or "nonresponsive" mice of: (a) aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity in liver, bowel, lung, kidney, and skin; (b) 7-ethoxycoumarin O-deethylase activity in liver and kidney; (c) hepatic p-nitroanisole O-demethylase and 3-methyl-4-methylaminoazobenzene Ndemethylase activities; and (d) the new formation of cytochrome P1-450 in liver, bowel, lung, kidney, and skin. Application of TCDD to the skin of either "responsive" or "nonresponsive" mice induces aryl hydrocarbon hydroxylase activity more than 6-fold in the skin and also in the liver. The inbred mice used in this study include the "responsive" C57BL/6N, C57BL/6J, C3H/HeN, BALB/cAnN, and CBA/HN strains and the "nonresponsive" DBA/2N, DBA/2J, AKR/N, NZW/BLN, and NZB/BLN strains. These data demonstrate that genetically "nonresponsive" mice have the structural and regulatory genes necessary for expression of these inducible microsomal monooxygenase activities and associated new formation of cytochrome P1-450 and that the defect in these mice is a failure to recognize aromatic hydrocarbons which are less potent inducers. In the "nonresponsive" inbred strains, perhaps a mutation has occurred which results in production of an inducerbinding receptor having a diminished affinity for aromatic hydrocarbons.

Pregnenolone-16α-carbonitrile (PCN), a potent catatoic steroid without known classical hormonal effects, was administered per os to female rats. Its effects were studied on mixed function oxygenases and on various phosphatases in liver microsomal subfractions: rough microsomes, smooth I, and smooth II microsomes. For comparison, phenobarbital (PB) and 3-methylcholanthrene (MC) were also administered. The inducers increased the protein content in total, rough and smooth I microsomes in the following order: PB, PCN, and MC, whereas the protein amount in smooth II microsomal fraction remained unchanged. The content of cytochrome P-450 was about doubled in all subfractions except the smooth II membranes, following treatment with any of the inducers. PCN differed in inducing specificity from PB in increasing benzo(α)pyrene hydroxylase and from MC in stimulating aminopyrine demethylase in total microsomes. PCN also differed from PB in enhancing the capacity not only for rough and smooth I microsomes, but also for smooth II microsomes to demethylase aminopyrine. No major difference in magnitude of effects among the inducers was noted between rough and smooth I microsomes. In contrast to the altered substrate specificities produced in the monooxygenase system by different inducers, a more uniform pattern of specificities was seen in microsomal phosphatases. PCN, MC and PB all increased the activities of nucleoside diphosphatase (IDPase), whereas G6Pase and ATPase activities were little affected. Cycloheximide was partially effective in depressing the increase of both the monooxygenase system (cytochrome P-450 and benzo(α)pyrene hydroxylase) and nucleoside diphosphatase activity. We conclude that (1) treatment with PCN results in different substrate specificity when compared to PB and MC; that (2) PCN is the most potent inducer of the three in stimulating drug hydroxylation in smooth II microsomes, that (3) various smooth microsomal membranes of liver cells are affected differently by inducers of drug metabolism, and finally (4) that drugs also alter some microsomal phosphatase activities but not all.

A microsomal mixed function oxidase isolated from V. rosea seedlings was shown to catalyze the hydroxylation of the monoterpene alcohols, geraniol and nerol, to their corresponding 10-hydroxy derivatives. Hydroxylase activity was dependent upon NADPH and oxygen and was associated with the 100,000 X g pellet which exhibited a characteristic reduced P-450-CO binding spectra. Light reversible inhibition by CO as well as differential sensitivity to other inhibitors established the hydroxylase as a cytochrome P-450 type. Cis-trans isomerase activity was not observed in this preparation. Both geraniol and nerol were shown to be hydroxylated almost exclusively at the C-10 methyl group.

The cytochrome P-450K containing monooxygenase system of rat kidney cortex microsomes catalyzes the hydroxylation of various saturated fatty acids of medium chain length to the corresponding ω- and (ω-1)-hydroxy derivatives. The hydroxylation activity, as well as the ratio between the two hydroxylated products, vary with the carbon chain length of the fatty acid. Optimal hydroxylation activity is observed with myristic acid which yields the 13- and 14-hydroxylated products at a ratio of about 1. The ω/(ω-1)-hydroxylation ratio decreases with increasing carbon chain length of the fatty acid. On the other hand, with lauric acid as a substrate the ratio between ω- and (ω-1)-hydroxylation does not change significantly with varying time of incubation or substrate concentration, or incubation in a medium containing D2O or after induction of enhanced hydroxylation activity by starvation of the animals. Furthermore, 12-hydroxylauric acid and capric acid—which is almost exclusively ω-hydroxylated by rat kidney cortex microsomes—inhibit both 11- and 12-hydroxylation of lauric acid to a similar extent whereas 11-hydroxylauric acid does not seem to inhibit either 11- or 12-hydroxylation. C10-C16 fatty acids produce the type I spectral change upon addition to rat kidney cortex microsomes and seem to interact with similar amounts of the cytochrome P-450K present in these particles. In agreement with the metabolic studies, 12-hydroxylauric acid interacts with cytochrome P-450K giving rise to a reverse type I spectral change, whereas 11-hydroxylauric acid does not produce an observable spectral change. Finally, results of binding experiments with a series of derivatives of dodecane suggest that type I binding to cytochrome P-450K requires, besides a proper chain length, the presence of a carbonyl group together with an electron pair on a neighboring atom at the end of the carbon chain. A chain length of 14 carbon atoms seems to be optimal and it is suggested that this chain length may correspond to the distance between a possible binding site and the catalytic site of cytochrome P-450K

Imidazole acetate monooxygenase, which catalyzes the hydroxylation of imidazole acetate, was purified about 250-fold from extracts of induced cells of a pseudomonad and was crystallized. The molecular weight was determined to be approximately 90,000, and the molecular activity was calculated to be 2,260 at 24°. The enzyme was shown to be homogeneous upon acrylamide gel electrophoresis and ultracentrifugation. The crystalline enzyme showed an absorption spectrum characteristic of a flavoprotein with absorption maxima at 270, 383, and 442 mµ. The flavin component of the enzyme was isolated and identified as flavin adenine dinucleotide, 1 mole of which was found per mole of enzyme. Aerobic incubation of the enzyme with imidazole acetate and reduced nicotinamide adenine dinucleotide resulted in the accumulation of an unstable intermediate having an absorption spectrum with a maximum at 259 mµ, which was indistinguishable from that of imidazolone acetate. Flavin bound to the enzyme was reduced by the anaerobic addition of a stoichiometric amount of reduced nicotinamide adenine dinucleotide. The reduced form of enzyme-bound flavin was found to serve as an electron donor for the hydroxylation reaction. Some other properties of the enzyme are also described.

A discussion of "oxygenases" or enzymes which are involved in the biological fixation of molecular oxygen points out that biosynthesis of prostaglandins from unsaturated fatty acids is catalyzed by the action of 2 successive reactions involving a dioxygenase and a monooxygenase.

In view of the observations in man that chronic marihuana smokers exhibit a reduction in the half-life of administered radioactively labeled tetrahydrocannabinol (Δ1-THC), the possibility that exposure to marihuana stimulates the hepatic metabolism of Δ1-THC was considered. Thus, the effects of Δ1-THC or of a marihuana extract, containing 25% Δ1-THC, on the hepatic microsomal monooxygenase system in rats were examined. In addition, for comparison purposes, the effect of technical grade DDT on this enzyme system was also studied. The intraperitoneal administration of Δ1-THC for 3–9 days or the oral administration of Δ1-THC for 3 days did not affect the activity of the in vitro microsomal demethylation of aminopyrine and p-chloro-N-methylaniline (PCMA) or the oxidative metabolism of [ 14 C ]Δ 1 - THC (determined as residual Δ1-THC or as metabolite-7-hydroxy-Δ1-THC). Similarly the oral administration of a whole marihuana extract for 7 days did not affect the in vitro demethylation of aminopyrine or the hydroxylation of Δ1-THC. On the other hand, intraperitoneal injections of DDT for 3 days increased about two-fold the oxidative metabolism of Δ1-THC, demonstrating that the microsomal oxidation of Δ1-THC can be enhanced by an inducer of the monooxygenase system.

Es wurde eine Pseudomonas-Spezies isoliert, die auf n-Butan und n-Pentansulfonat als Kohlenstoffquelle ein gutes Wachstum zeigte. Der Abbau dieser Verbindungen verlauft uber eine Aufspaltung der C-S-Bindung. Zusatzliche Abbauwege konnten ausgeschlossen werden. Mit rohen Enzymextrakten konnte gezeigt werden, das das sulfonatspaltende Enzym in seiner Wirkung vom freien Sauerstoff abhangig ist und durch NAD(P)H stimuliert wird. Dies deutet auf eine hydroxylierende Monooxygenase hin. Eine geringe Menge Aldehyd und das Sulfit-Ion konnten nachgewiesen werden. Diese Ergebnisse zeigen, das Alkan-1-hydroxy-1-sulfonat das erste Zwischenprodukt ist. Microbiological Degradation of n-Alkane-1-Sulfonates A Pseudomonas species which grows well on n-butane- and n-pentanesulfonate as the source of carbon has been isolated. The aforesaid compounds are degraded only via splitting of the C-S bond. Using crude enzyme extracts it has been shown that the action of the enzyme responsible for splitting the sulfonates is dependent on free oxygen and is promoted by NAD(P)H. This indicates the presence of a hydroxylating monooxygenase. A minute amount of aldehyde and sulfite ions has been detected. These results indicate that alkane-1-hydroxy-1-sulfonate is the first intermediate product.

Both the rat kidney cortex aryl hydrocarbon hydroxylase activity and cytochrome P-450 K are induced by benzo(α)pyrene treatment. Following a single injection of benzo(α)pyrene, maximal hydroxylase activity and cytochrome P-450 K content occur at 24 hr, returning to control levels within 72–96 hr. Induction of both the enzyme activity and hemoprotein is inhibited by cycloheximide. The enzyme system is localized in the microsomal fraction, has an absolute requirement for NADPH and molecular oxygen, and a pH optimum at 7.4; the induced activity is linear with microsomal protein concentration up to 0.8 mg and with time up to 20 min. Both the hydroxylase activity and cytochrome P-450 K follow the same pattern of inactivation with increasing temperature. The apparent K m for the induced hydroxylase was 7.7 μ m and V was increased fourfold above control value. In the presence of laurate, a substrate for the kidney microsomal cytochrome P-450 K -dependent monooxygenase system, the amount of inhibition of hydroxylase activity corresponded to the level of activity present in untreated kidney cortex microsomes. α-Naphthoflavone (10 −5 m ), a type I inducer (36) produced a greater inhibitory effect on the induced hydroxylase activity than on the control (55% vs 20%). The presence of cytochrome c or carbon monoxide markedly decreased hydroxylase activity. This evidence in addition to aforementioned characteristics of the enzyme suggests a cytochrome P-450 K -dependent aryl hydroxylase activity which differs from that present in the control rat.

An inducible oxidase from Arthrobacter oxydans that catalyzes the oxidation of 2,6-dihydroxypyridine was purified about 50-fold. Reduced pyridine nucleotide is required for oxidase activity; no requirement for ferrous, ferric, or other cation could be demonstrated. Artificial electron acceptors do not substitute for oxygen in the reaction. The oxidase catalyzes the anaerobic reduction of methylene blue by NADH. 2,6-Dihydroxypyridine increases the rate of the anaerobic reaction but is not altered during the reaction. The purified oxidase preparation has absorption maxima at 384 and 455 nm. The maxima disappear during the anaerobic reaction. These and other data suggest that the oxidase is a monooxygenase of molecular weight about 89,000 having a flavin prosthetic group. The only detectable product of the oxidase reaction is a blue pigment. Its formation is decreased or eliminated when oxidase is supplemented with a second enzyme fraction (cleavage enzyme). Blue pigment is not a substrate for the cleavage enzyme. It is proposed that the normal metabolic sequence is the oxidation of 2,6-dihydroxypyridine to a trihydroxypyridine, mediated by the oxidase, followed by pyridine ring cleavage in a second reaction. The over-all stoichiometry of the oxidase reaction, corrected for oxygen utilized in blue pigment formation, is 2,6-dihydroxypyridine:NADH:O2 = 1:1:1.

Rat liver microsomes previously heated to 50° for 5 min accumulate 2,3-oxidosqualene on incubation with squalene. Squalene epoxidase activity can be assayed either with squalene and heated microsomes or with 10,11-dihydrosqualene and intact microsomes. In common with other monooxygenases, the epoxidase requires TPNH and molecular oxygen. Both a soluble fraction of rat liver and microsomes are necessary for enzyme activity. Carbon monoxide or potassium cyanide fail to inhibit squalene epoxidation.

Aquayamycin inhibits partially purified tryptophan 5-monooxygenase of guinea-pig brainstem about 40 and 80% at 0.1 and 1 μM respectively. The inhibition is not competitive with respect to substrate, DMPH4∗∗ or Fe++ but is reversed by DTT∗∗. Tryptophan 2,3-dioxygenase was also inhibited almost completely at 2 μM, but other oxygenases including metapyrocatechase, protocatechuate 3,4-dioxygenase, lysine monooxygenase and imidazoleacetate monooxygenase were not inhibited at all at 1 μM.

1. 1. n-Heptane was used as substrate for the monooxygenase system in rat liver microsomes. All isomeric alcohols were found as hydroxylation products, 2-heptanol being the main product. 2. 2. Phenobarbital treatment of rats caused a 4-fold increase in the formation of 2-, 3- and 4-heptanol but only a small enhancement in the formation of 1-heptanol. 3,4-Benzpyrene pretreatment of rats did not significantly change the specific hydroxylation activity but led to a complete change in the pattern of the isomeric alcohols formed. 3. 3. Inhibition by CO and metyrapone affected the ω- and (ω − 1)-hydroxylation differently. 4. 4. It can be concluded that at least three monooxygenase activities are involved in the microsomal hydroxylation of n-heptane.

Crystalline l-lysine monooxygenase from Pseudomonas fluorescens catalyzes the incorporation of 1 atom of molecular oxygen into the substrate, l-lysine, concomitant with a decarboxylation to form δ-amino-n-valeramide. In addition to l-lysine, the l isomers of several basic amino acids such as 2,7-diaminoheptanoate, arginine, δ-hydroxylysine, and S-aminoethylcysteine serve as the substrate for the monooxygenation catalyzed by the enzyme. When l-ornithine was incubated with the enzyme, however, it was converted to α-keto-δ-amino-n-valerate with the consumption of oxygen and with the formation of ammonia and hydrogen peroxide in stoichiometric amounts. These findings suggest that l-lysine monooxygenase can catalyze an oxidative deamination of l-ornithine similar to that catalyzed by l-amino acid oxidase. 2,8-Diaminooctanoate appeared to be degraded in the same way as l-ornithine as evidenced by a 50% reduction in the rate of oxygen uptake upon the addition of catalase. Evidence indicating that both activities, monooxygenation and oxidation of amino acids, are attributable to a single enzyme includes the constant ratio of the two activities during the course of the enzyme purification and the parallel inactivation by —SH inhibitors or by sodium dodecyl sulfate. A competitive type inhibition of lysine monooxygenation by ornithine suggests that both amino acids bind to the enzyme at the same site.

Rapidly sedimenting CaCl2-aggregated microsomes from rat liver were found to catalyze the demethylation of aminopyrine (AP) and p-chloro-N-methylaniline (PCMA) and the hydroxylation of Δ1-tetrahydrocannabinol (Δ1-THC). The demethylation of AP required NADPH and O2 and was inhibited by carbon monoxide. This enzymatic activity was stimulated by the prior treatment of immature male rats with phenobarbital. The specific enzymatic activity of the CaCl2-aggregated microsomes was similar to that obtained with normal (100,000 g centrifuged) microsomes. It is suggested that the CaCl2-aggregation yields functionally-intact microsomes.

Enterotoximia in dyspepsia is caused by the microorganisms toxins of microbial association and biogenic amines formed in the intestinal lumen, as a result of a violation of not only cavity, but also parietal digestion. Liver damage when exposed to various toxicants is associated with the metabolism and activity of cytochrome P450, an indicator of the monooxygenase system. In recent decades, the attention of scientists has been receiving the problem of medicinal liver damage. "Startin-phyto" is a combined preparation aimed to removing endogenous intoxication in the treatment of dyspepsia in farm animal young stock. The effect of the drug on monooxygenase and antitoxic liver functions was studied using D.G. Rosin method (1964). It is based on the ability of various chemicals to affect amounts sleep of laboratory rodents (mice, rats) caused by hexenal, which is known to be inactivated in the liver. Five groups of two-month-old female rats were assembled. The animals of the experimental groups (15 animals) have been injected with "Startin-phyto" orally one time at a dose of 6.3 ml / kg. Distilled water for the control group (6 animals) has been used. The general condition of animals was characterized by the biochemical parameters of the blood: the amount of total protein, urea, glucose, total bilirubin, creatinine and the activity of the enzymes aspartate and alanine aminotransferase, lactate dehydrogenase. Total protein was analyzed by biuret reaction; glucose – by the unified glucose oxidase method for orthotolidine oxidation; the creatinine concentration in the blood serum of calves – in according to the Jaffe color reaction unified method; urea – by the urease method. The carried out complex experimental studies have shown that the drug "Startin-phyto", when administered once to rats, enhances detoxification processes in the liver, is safe and does not have a toxic effect on the liver.

The binding of metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone) by Pseudomonas putida cytochrome P-450 is described. The absolute absorption spectrum of the metyrapone-cytochrome P-450 complex in the ferric form has absorption maxima at 421 and 536 nm. In the ferrous form, this complex has absorption maxima at 442, 539, and 566 nm. The equilibrium constant for the binding of metyrapone by oxidized cytochrome P-450 is 2.3×108 m −1 and the binding is competitive with the substrate camphor. Metyrapone inhibits the uptake of oxygen by the coupled monooxygenase system probably by competing with camphor for the camphor-binding site on oxidized cytochrome P-450. The results presented indicate that the camphor-binding site is close to the oxygen-activating site on cytochrome P-450.

The relative proportions of high spin (having electron paramagnetic resonance signals at g = 8.0, 3.7, and 1.7 when measured below 10°K) and low spin (having signals at g = 2.4, 2.3, and 1.9 when measured at 77°K) cytochrome P450 iron are different when liver microsomes from control rabbits, rats, and mice are compared with those from phenobarbital-treated animals. Treatment of rabbits, rats, or genetically “responsive” mice with 3-methylcholanthrene, however, causes an increase principally in the g = 8.01 signal height and little or no change in the g = 2.27 signal intensity. Contrary to what had been found in the mouse, the 3-methylcholanthrene-inducible aryl hydrocarbon hydroxylase, 7-ethoxycoumarin O-deethylase, p-nitroanisole O-demethylase, and 3-methyl-4-methyl-aminoazobenzene N-demethylase activities are not as closely associated with the same genetic region in the rat and with increases in the g = 8.01 signal height in either the rat or rabbit. In rabbit or mouse microsomes treated in vitro with varying amounts of sodium cholate or potassium thiocyanate and dialyzed or not dialyzed, a g = 5.95 signal is always associated with cytochrome P420 concentration, indicating that this signal probably represents the low field g tensor of high spin P420 iron. In the rabbit, an initial rise in the g = 8.01 signal height occurs in the first 2 hours after aromatic hydrocarbon administration, followed by a secondary increase in the g = 8.01 signal height between 6 and 24 hours; in the rat or genetically responsive mouse, only this latter secondary rise is seen. These results suggest that the initial rise represents binding of 3-methylcholanthrene or other substrate to cytochrome P450 and that the secondary rise probably reflects a genetically mediated process, e.g. a new membrane protein or lipid being formed or a membrane configurational change concomitant with the formation of cytochrome P448. Under a variety of experimental conditions including microsomes from 3-methylcholanthrene-treated genetically responsive and “nonresponsive” mice, the dithionite reduced minus oxidized difference in absorbance between 645 and 700 nm at room temperature correlates well with the g = 8.01 signal height measured below 10°K.

Evidence for mono-oxygenase activity during the metabolism of thioethers by the fungus Aspergillus niger is presented. Attempts to obtain selenoxides as microbial metabolic products are described.

Prostaglandin E1 was synthesized by incubating 81114-eicosatrienoic acid with a proportion of vesicular gland for 10 minutes in an atmosphere of labeled oxygen. To determine the origin of the oxygens of the ring a derivative was oxidized by permanganate-periodate and analyzed by mass spectrometry. The findings show that the oxygen atoms of the hydroxyl group at C-11 and of the keto group at C-9 originate in the same molecule of oxygen. Two distinct reactions are involved in this transformation: a dioxygenase and a monooxygenase reaction. Dioxygenation probably occurs by the addition of oxygen across carbons 9 and 11 with concomitant ring closure between carbons 8 and 12 involving a conrotatory process. Conversion to prostaglandin E1 also involves isomerization of a double bond and introduction of a hydroxyl group at C-15. Another mechanism involved may be the formatiom of a hydroperoxide at C-11 with isomerization of a double bond. Further studies should be done to determine whether or not the introduction of the oxygens at C-9 and C-11 precedes the introduction of oxygen at C-15.

A polychlorinated biphenyl (PCB) compound, Clophen A 50, enhanced both hepatic aryl hydrocarbon hydroxylase and p-nitroanisole O-demethylase activities (7.5-fold and 16-fold, respectively), after treating the rats for 6 days with consecutive daily injections of Clophen A 50 (15 mg/kg i.p.). The treatment increased 3-fold the content of the carbon monoxide binding hemoprotein in liver microsomes, causing a concomitant shift in its reduced carbon monoxide absorbance peak to 448 nm. NADPH cytochrome c reductase, another component reaction of the microsomal mixed-function oxidase, was enhanced 1.5-fold in 6 days. A slight enhancement in the overall hydroxylation reactions was already observable 24 h after a single injection of Clophen A 50. The UDPglucuronosyltransferase activity of native liver microsomes was enhanced 3-fold in 6 days by the Clophen A 50 treatment of rats. The enhancement was, however, more pronounced, if the microsomes were treated in vitro with membrane-perturbing agents to activate the latent UDPglucuronosyltransferase before measuring its activity. After treatment for 6 days, the enhancement was about 6-fold in digitonin-treated, 5-fold in phospholipase C-treated and about 10-fold in trypsin-digested microsomes. No enhancement could be detected 24 h after a single Clophen A 50 injection. Aryl hydrocarbon hydroxylase activity was also enhanced in lung (5-fold), and kidney (8-fold) microsomes, whereas the microsomes from the duodenal mucosa exhibited no enhancement by a Clophen A 50 treatment of rats for 3 days. The data obtained support the assumption that PCBs form a new type of inducer group in enhancing the microsomal drug biotransformation. Both the monooxygenase complex and UDPglucuronosyltransferase differ in their properties from those after enhancement with the known types of inducers, exemplified by phenobarbital and 3-methylcholanthrene, respectively.

The enzymatic hydroxylation reactions of flavin monooxygenases are suggested to involve an oxaziridine as the active oxygenating agent. The chemistry of these monooxygenases and their nonenzymatic model systems are consistent with an oxaziridine intermediate. Several advantages of the oxaziridine model over the previously proposed “carbonyl oxide” and flavin peroxide models are discussed.

The spectral changes associated with the addition of prostaglandins (PGs) to hepatic microsomes from guinea pigs and rats were examined. PGA 1 , PGA 2 , PGE 1 , PGE 2 , PGF 1α and PGF 2α when added to guinea pig liver microsomes exhibited type I spectra. The binding affinities as determined from spectral dissociation constants (K s ) were highest with PGA 1 and PGA 2 . With liver microsomes from control or 3-methyl-cholanthrene (MC)-treated rats, PGs did not yield type I spectra; however, in this case a weak spectrum, designated here as type “II” was at times observed, With microsomes from phenobarbital (Pb)-treated rats only PGA 1 and PGA 2 yielded type I spectra; again in absence of type I spectrum, a weak type “II” was occasionally observed. The addition of PGA 1 and PGA 2 to liver microsomes from Pb-treated rats inhibited the microcomal mediated hydroxylation of hexobarbital. The inhibition by PGA 1 was competitive; the K i = 8.2 × 10 −4 M was found to be similar in magnitude to the K s = 7.3 × 10 −4 M of PGA 1 observed with rat liver microsomes. These observations suggested that PGs particularly of the A series interact with the hepatic microsomal cytochrome P-450 monooxygenase system.

A single dose of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) (160 mg/kg i.p.) enhanced the monooxygenase step of drug biotransformation in rat liver. The O-demethylation of p-nitroanisole was especially increased, a peak in activity approximately 5-fold compared with controls being attained in 7 days. On the other hand, there was only a 2-fold increase in aryl hydrocarbon hydroxylase activity. DDT increased the cytochrome P-450 content of the liver, this increase coincided well with that in p-nitroanisole O-demethylation activity. The UDPglucuronosyltransferase activity of liver microsomes was not enhanced by DDT administration, unless the microsomes were pretreated to reveal latent activity prior to assay. After trypsin digestion of microsomes a maximum increase in activity of approximately 3-fold was observed as a result of DDT dosage. The canonic surfactant cetylpyridinium chloride was less active in revealing the latent UDP-glucuronosyltransferase activity, and two other membrane perturbants, the detergent digitonin and phospholipase A, were unable to show enhancement in UDPglucuronosyltransferase as a result of DDT dosage.

When mice are continuously exposed to an atmosphere containing 2.5–3% n -hexane over a period of 4 days liver growth is observed after 24 h. Concomitantly the specific activities of cytochrome P450 and the cytochrome P450 reductase increase with a maximum after 38 h. The cytochrome b 5 levels in liver microsomes begin to rise at the second day and continue to increase even after the fourth day. No strict correlation is observed between the cytochrome P4SO content and the monooxygenation activity for cyclohexane. The pattern of hydroxylation products from n -hexane changes significantly upon pretreatment of the animals, suggesting that a qualitative alteration in the cytochrome P450 species had occurred.

The potential for selected microorganisms to hydroxylate aromatic substrates in a manner analogous to mammalian systems has been studied. Based on literature precedence and prior experience, 11 microorganisms were chosen from among a variety of genera of fungi and bacterial species and were incubated with 13 model compounds including acetanilide, acronycine, aniline, anisole, benzene, benzoic acid, biphenyl, chlorobenzene, coumarin, naphthalene, nitrobenzene, trans -stilbene, and toluene. In most instances, the microbial model system yielded patterns of phenolic metabolites similar to those reported with cytochrome P 450 monooxygenases of hepatic microsomes and/or in vivo mammalian systems. Furthermore, N -acetylation of aniline, N -deacetylation of acetanilide, and O -demethylation of anisole were found with certain organisms. The potential usefulness of microbial systems for the synthesis of preparative quantities of mammalian metabolites of foreign organic compounds is discussed.

4-hydroxybenzoate hydroxylase from Ps. putida has been studied with regard to the oxygen interaction with the reduced flavin. By rapid scanning spectrophotometry and EPR-measurements of rapidly frozen mixtures no intermediate could be established either in the presence of 4-hydroxybenzoate or the dead-end inhibitor 6-hydroxynicotinic acid.

Publisher Summary This chapter discusses about substrate interaction with microsomal cytochrome P-450. Cytochrome P-450 is the terminal oxidase involved in a number of NADPH-linked microsomal hydroxylation (monooxygenase) reactions. The spectral studies on the interaction of various substrates with microsomal cytochrome P-450 are reported. Imipramine is a tricyclic compound, which upon incubation with rat-liver microsomes in the presence of NADPH and O 2 gives rise to the formation of 2-hydroxyimipramine and desmethylimipramine (DMI). The type I spectral change obtained with imipramine, DMI and 2-OH-DMI reflects the interaction of the compounds with the same cytochrome P-450 species is shown by the experiments. Evidence for the relationship between the type I spectral change and oxidative metabolism comes from the finding that the calculated binding constants of various substrates parallel their apparent Michaelis constants.

Human fetal liver microsomes were shown to contain the components of the NADPH- and NADH-linked electron transport systems. The NADPH-linked system was capable of hydroxylating testosterone and laurate, but unable to oxidize exogenous substrates such as 3, 4-benzpyrene and aminopyrine. The spectral change associated with the binding of the substrate to cytochrome P-450 was of the type I variety with testosterone and laurate and type II with aminopyrine.

Publisher Summary This chapter discusses the methods of preparation of Imidazoleacetate Monooxygenase ( Pseudomonas ). The enzyme activity is assayed by measuring the decrease in optical density at 340 mμ because of the substrate-dependent oxidation of NADH. Alternatively, the activity may be assayed polarographically by measuring the oxygen consumption. One unit of enzyme is defined as that amount that causes the oxidation of 1 micromole of NADH per minute at 24°; the specific activity is defined as units per milligram of protein. The enzyme activity is markedly inhibited by silver and mercury compounds, although iodoacetamide and iodoacetate show much less effect. Metal chelating agents, including o -phenanthroline, α,α'-dipyridyl, and 8-hydroxyquinoline do not show appreciable inhibitory activity. The enzyme contains 1 mole of FAD per mole of enzyme, the molecular weight of which is estimated to be approximately 90,000. Metal components, such as iron or copper, are not detected; they appear to be absent or present only in insignificant amounts.

The postulated biosynthetic pathway (Blaschko, 1939) for the formation of the adrenergic transmitters (Fig. 1) was the subject of several studies and was confirmed with the use of radioactive labeled precursors. Three enzymes are involved in the catalysis of norepinephrine biosynthesis: tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and dopamines-hydroxylase (DβH; a fourth enzyme, namely, phenylethanolamine-N-methyltransferase (PNMT) catalyzes the biosynthesis of epinephrine from norepinephrine. Two enzymes, namely, TH and DβH, can be classified as mixed-function oxidase (Mason, 1957) or monooxygenase (Hayaishi, 1964). The general reaction catalyzed by monooxygenases is shown in Reaction (1) where RH stands for the substrate to be hydroxylated, ROH for the hydroxylated product, and XH2 for the electron donor.

The TPNH dependent monooxygenases, which are localized in the endoplasmic reticulum of the liver cell, have been implicated in the majority of drug oxidations. In addition to these enzymes, however, there are a number of other classes of enzymes which are involved in the oxidation and reduction of foreign chemicals in the body. These include among others the oxido-reductases as well as those enzymes responsible for the oxidation of aldehydes to carboxylic acids. The oxidoreductases are a class of dehydrogenases that catalyze the conversion of alcohols to aldehydes or ketones or alternatively the reduction of carbonyl compounds to alcohols.

The interaction of various substrates with the cytochrome P-450 containing monooxygenase system of rat liver microsomes has been studied. Testosterone, triamcinolone acetonide (TrA) and laurate were found to interfere with the type I spectral changes produced by the addition of hexobarbital and ethylmorphine to suspensions of microsomes suggesting that these substrates bind to a common cytochrome P-450 species. Substrates producing a type I spectral change with liver microsomes, such as ethylmorphine, hexobarbital, aminopyrine, laurate and testosterone enhanced the rate of NADPH-linked cyto-chrome P-450 reduction. TrA, on the other hand, did not elicit a type I spectral change and did not stimulate the rate of reduction of the cytochrome. However, TrA interfered with the stimulation of this rate by hexobarbital and aminopyrine. Furthermore, when two compounds which stimulate the rate of cytochrome P-450 reduction were present together, there were no additive effects in stimulation of this rate. In fact, there was a diminution of the stimulation observed when the more potent of the two was present alone. The results suggest that the substrates of the liver-microsomal monooxygenase system studied interact with a single cytochrome P-450 species and that the competitive inhibition they exert on each other's metabolism may be related to an interference with the binding to cytochrome P-450.