Pseudomonas putida A ATCC 12633 oxidizes trimethylamine aerobically via two different pathways

The present study examined the aerobic metabolism of trimethylamine in Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine. In both conditions, the trimethylamine was used as a nitrogen source and also accumulated in the cell, slowing the bacterial growth. Decreased bacterial growth was counteracted by the addition of AlCl₃. Cell-free extracts prepared from cells grown aerobically on tetradecyltrimethylammonium bromide exhibited trimethylamine monooxygenase activity that produced trimethylamine N-oxide and trimethylamine N-oxide demethylase activity that produced dimethylamine. Cell-free extracts from cells grown on trimethylamine exhibited trimethylamine dehydrogenase activity that produced dimethylamine, which was oxidized to methanal and methylamine by dimethylamine dehydrogenase. These results show that this bacterial strain uses two enzymes to initiate the oxidation of trimethylamine in aerobic conditions. The apparent K_m for trimethylamine was 0.7 mM for trimethylamine monooxygenase and 4.0 mM for trimethylamine dehydrogenase, but both enzymes maintain similar catalytic efficiency (0.5 and 0.4, respectively). Trimethylamine dehydrogenase was inhibited by trimethylamine from 1 mM. Therefore, the accumulation of trimethylamine inside Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine may be due to the low catalytic efficiency of trimethylamine monooxygenase and trimethylamine dehydrogenase.     

A novel denitrifying bacterial isolate that degrades trimethylamine both aerobically and anaerobically via two different pathways

The aerobic and anaerobic degradation of trimethylamine by a newly isolated denitrifying bacterium from an enrichment culture with trimethylamine inoculated with activated sludge was studied. Based on 16S rDNA analysis, this strain was identified as a Paracoccus sp. The isolate, strain T231, aerobically degraded trimethylamine, dimethylamine and methylamine and released a stoichiometric amount of ammonium ion into the culture fluid as a metabolic product, indicating that these methylated amines were completely degraded to formaldehyde and ammonia. The strain degraded trimethylamine also under denitrifying conditions and consumed a stoichiometric amount of nitrate, demonstrating that complete degradation of trimethylamine was coupled with nitrate reduction. Cell-free extract prepared from cells grown aerobically on trimethylamine exhibited activities of trimethylamine mono-oxygenase, trimethylamine N-oxide demethylase, dimethylamine mono-oxygenase, and methylamine mono-oxygenase. Cell-free extract from cells grown anaerobically on trimethylamine and nitrate exhibited activities of trimethylamine dehydrogenase and dimethylamine dehydrogenase. These results indicate that strain T231 had two different pathways for aerobic and anaerobic degradation of trimethylamine. This is a new feature for trimethylamine metabolism in denitrifying bacteria.     

Adenosine 3′:5′-cyclic monophosphate in young and senescent human fibroblasts during growth and stationary phase in vitro. Effects of prostaglandin E1 and of adrenaline

1. A mono-oxygenase, which oxidizes trimethylamine and other tertiary amines bearing methyl or ethyl groups, was partially purified sixfold from Pseudomonas aminovorans grown on trimethylamine as sole carbon source. 2. The preferred electron donor was NADPH. The enzyme had a pH optimum of 8.0-9.4 for trimethylamine oxidation, and 8.8-9.2 for dimethylamine oxidation. 3. The oxidation product of trimethylamine was shown to be trimethylamine N-oxide. Other tertiary amines were probably also converted into N-oxides. 4. The enzyme also oxidized secondary amines. 5. The oxidation of trimethylamine was only slightly inhibited by CO and not at all by KCN or proadifen hydrochloride (SKF 525-A), but was inhibited by trimethylsulphonium chloride, tetramethylammonium chloride, 2,4-dichloro-6-phenylphenoxyethylamine (Lilly 53325) and its NN-diethyl derivative (Lilly 18947). 6. The oxidation of dimethylamine showed a similar response to inhibitors and a parallel loss in activity on heating at 35 degrees C. 7. The activities of the trimethylamine mono-oxygenase, trimethylamine N-oxide demethylase and the secondary-amine mono-oxygenase increased severalfold during adaptation of succinate-grown bacteria to growth on trimethylamine, and the trimethylamine mono-oxygenase was the first enzyme to show an increase in activity. It is concluded that all three enzymes are involved in growth on trimethylamine by this organism.     

Reduced glutathione inhibits the alkylation by N-nitrosodimethylamine of liver DNA in vivo and microsomal fraction in vitro

The transfer of radioactivity from N-nitroso-[14C]dimethylamine to trichloroacetic acid precipitable macromolecules in the microsomal fraction of rat liver was investigated. This transfer was found to depend on N-nitrosodimethylamine being metabolized. Cytosolic fraction and cytosol enriched with reduced glutathione inhibited the binding of radioactivity to acid insoluble proteins. Depletion of glutathione in rat liver with diethylmaleate prior to i.v. administration of 10 mg N-nitroso-[14C]dimethylamine/kg led to an increase in O6-methylguanine and N-7-methylguanine in DNA. If rats were fed disulfiram for 6 days (2 g/kg feed), glutathione and glutathione S-transferase were enhanced, and the degree of methylation of guanine by N-nitrosodimethylamine was greatly reduced, as was the metabolism of N-nitrosodimethylamine in the intact animal. Fasting rats for 24 h did not change the N-nitrosodimethylamine-demethylase activity in vitro but greatly enhanced the methylation of guanine in vivo, while the glutathione content and glutathione S-transferase activity were not changed compared to fed animals.     

The light cycle as a modulator of aminopyrine demethylation by liver from normal,orchiectomized or adrenalectomized rats

Abstract

As part of studies on the interactive effects of light and endocrine factors on hepatic mixed‐function oxidases, the aminopyrine demethylase activity of liver microsomes and the serum concentrations of corticosterone and testosterone were measured in adult male rats (sham‐operated, castrated or adrenalectomized) exposed for 21 days to one of the following types of environmental lighting: (1) Normal light cyle (L from 09.00 to 21.00 h); (2) Reverse light cycle; (3) Constant light (LL) or (4) Constant darkness (DD). One half of the animals in each of the 12 groups was killed at 06.00 h and the other at 18.00 h on the last day of treatment. A 3‐way analysis of variance, multiple comparisons and correlations allowed the following conclusions: (1) The type of lighting had the most significant effect on demethylase activity, its highest values corresponding to periods of darkness (particularly in the rats exposed to a reverse light cycle) and its lowest to LL. (2) Castrated rats as a group had the lowest demethylase activity. (3) Time of death was a significant factor, particularly for the rats in a reverse light cycle (higher activity at 18.00 than at 06.00 h). (4) There was a significant and positive correlation between aminopyrine demethylase and the rates of 7α‐hydroxylation of 3β‐hydroxy‐5‐androsten‐17‐one previously reported for the same animals. (5) There was no significant correlation between demethylase activity and serum corticosterone or testosterone.     

Alterations of liver ergastoplasmic membranes during DL-ethionine hepatocarcinogenesis. Aminopyrine demethylase activity and ribosome-membrane interaction

The activity of microsomal aminopyrine demethylase and the degree of association between ribosomes and endoplasmic membranes were studied in liver of ethionine-fed rats, as well as in hyperplastic nodules and in hepatoma. In the course of the ethionine-feeding, inhibition of aminopyrine demethylase and increase in the relative amounts of membrane-free ribosomes occured in liver cells. After 1–3 months from the end of the ethionine feeding, when hyperplastic nodules and hepatoma develop in the liver, a sharp inhibition of aminopyrine demethylase was found in the latter tissues, but not in the surrounding nonnodular liver. At the same time an increase of membrane-free ribosomes occured in surrounding nonnodular liver, in nodules and in hepatoma. The extent of this alteration, however, was significantly lower in surrounding nonnodular liver than in nodules and in hepatoma. These results are discussed in relation to the problem of the cellular precursors of hepatoma.     

Mechanism of induction of cytochrome P-450ac (P-450j) in chemically induced and spontaneously diabetic rats

Previous studies demonstrated that a microsomal high-affinity N-nitrosodimethylamine demethylase activity and cytochrome P-450ac (an acetone/ethanol-inducible form) were induced by streptozotocin-induced diabetes in rats. In the present work, the induction was studied in detail in two chemically induced (by streptozotocin and alloxan) diabetic rat models and one spontaneously (BB/Wor) diabetic rat model. All the diabetic conditions caused increases in three parameters: (a) microsomal N-nitrosodimethylamine demethylase activity which is known to be a good indicator of the level of P-450ac; (b) the levels of P-450ac as determined by immunoblot analysis; and (c) the levels of mRNA of P-450ac as determined by hybridization assays with a cDNA probe for this enzyme. These increases were abolished by treatment of the diabetic rats with insulin. The results suggest that the pathophysiological condition of diabetes is responsible for the induction of P-450ac and elevation of mRNA is involved in all of the three diabetic models investigated.     

Studies on the mechanisms of induction of N-nitrosodimethylamine demethylase by fasting,acetone, and ethanol

Previous work has shown that induction of a high-affinity NADPH-dependent nitrosodimethylamine demethylase (NDMAd) in liver microsomes occurs in rats due to fasting, ethanol consumption, and streptozotocin-induced diabetes. Several lines of observations suggest that this is due to the induction of specific cytochrome P-450 isozymes. Induction of P-450 species by ethanol has also been observed by other investigators. Since each of the above altered metabolic states has in common elevated levels of ketone bodies, the possible role of acetone, a known inducer of NDMAd, in the induction of the demethylase activity was investigated. Levels of endogenous acetone in fasted rats correlated (r = 0.72) with a three- to fourfold increase in NDMAd activity. However, a dose-response experiment showed endogenous levels of acetone to be capable of causing at most 40% of the induction in fasted rats. This suggests that other ketone bodies or factors may have contributed to the induction. The induction of NDMAd by ethanol was enhanced by alcohol dehydrogenase inhibitors pyrazole and acetaldehyde oxime, suggesting that ethanol, rather than its metabolites, was responsible for the induction.     

The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of S-Adenosylmethionine

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.     

Effects of pregnenolone-16 alpha-carbonitrile on drug metabolizing enzymes in hypophysectomized female rats

Treatment of intact and hypophysectomized female rats with pregnenolone-16 alpha-carbonitrile (PCN) resulted in a significant increase in hepatic aryl hydrocarbon hydroxylase (AHH) activity. However, the total cytochrome P-450 concentration, as measured by CO difference spectra, was increased to a greater extent in hypophysectomized rats than in intact rats. Total cytochrome P-450 was found to be 0.82 +/- 0.16 vs 2.43 +/- 0.31 nmoles/mg protein for control and PCN-treated hypophysectomized rats, respectively, and 0.68 +/- 0.23 vs 1.28 +/- 0.05 nmoles/mg protein for control and PCN-treated intact rats respectively. The concentration of metyrapone complex in microsomes from intact control and PCN-treated rats was found to be 0.4 +/- 0.11 vs 1.88 +/- 0.23 M respectively. Treatment of hypophysectomized rats with PCN resulted in an approximate 10-fold increase in the concentration of the metyrapone complex (0.42 +/- 0.15 M for control and 4.46 +/- 0.44 M for PCN-treated). Microsomal NADPH and NADPH cytochrome c reductase activities were also altered by PCN-treatment. Aminopyrine demethylase activity was stimulated approximately three-fold by PCN treatment in both intact and hypophysectomized rats. Benzphetamine demethylase activity was not significantly affected by PCN treatment. The results of these studies suggest that the absence of the pituitary gland can markedly influence PCN induction of cytochrome P-450 in the liver in female rats. PCN also differentially affects microsomal mixed-function oxidase activities associated with drug and xenobiotic metabolism.     

Effect of spermine on the inhibition by fatty acid on AMP deaminase reaction as a control system of the adenylate energy charge in yeast

Treatment of rats with pyrazole elevated the hepatic microsomal dimethylnitrosamine demethylase activity (DMNd) by several fold. Methylethylnitrosamine demethylase activity was also increased by pyrazole, but some classical monooxygenase activities were not induced. The treatment induced a new protein species which has an apparent molecular weight of 52,000 dal and is believed to be a cytochrome P-450 isozyme. The involvement of a hemoprotein in the pyrazole-induced DMNd was demonstrated in an experiment with CoCl₂ which decreased both the microsomal cytochrome P-450 content and DMNd. The induced enzyme with a single K_m value of 0.061 mM and V_max of 12.1 nmol/min/mg is probably the most efficient enzyme known to metabolize nitrosamines. NADPH-cytochrome P-450 reductase was also demonstrated to be an essential component enzyme of the DMNd. These results further substantiate the idea that the P-450-containing monooxygenase is responsible for the metabolism of dimethylnitrosamine in both the control and pyrazole induced microsomes.     

Immunochemical studies on the metabolism of nitrosamines by ethanol-inducible cytochrome P-450

The ethanol-induced rabbit liver microsomal cytochrome P-450, P-450LM3a, has been shown previously to efficiently catalyze the demethylation of N-nitrosodimethylamine (NDMA) with a Km of 2.9 mM. Since the predominant Km in hepatic microsomes from ethanol-treated rabbits is 0.07 mM, the role of P-450LM3a in the activation of this carcinogen has been uncertain. In the present study, antibodies to P-450LM3a were shown to almost completely inhibit NDMA demethylation by the purified P-450 in a reconstituted system as well as the low-Km activity of liver microsomes from control or ethanol-treated rabbits. In contrast, the antibody did not inhibit the high-Km NDMA demethylase activity in the microsomes. These results indicate that P-450LM3a is the major P-450 responsible for the low-Km NDMA demethylase activity. In addition, evidence is provided for the existence of a cytochrome immunochemically similar to P-450LM3a in liver microsomes from rats, mice, and guinea pigs that effectively catalyzes the demethylation of NDMA.     

Effect of denervation of the liver on the system of microsomal oxidation in adult and old rats

The role of adrenergic and cholinergic neural regulation in the functional activity of the liver microsomal oxidation enzymes has been studied. The experiments on adult and old rats using surgical denervation of the liver (vagotomy and sympathectomy++) have revealed changes in the monooxygenase activity (aminopyrine demethylase and aniline hydroxylase), in isoform composition and inductive synthesis of cytochrome P-450. The neural control over detoxication function of the liver is found to weaken in old age.     

The role of cytochrome P-450 in cholesterol biogenesis and catabolism

1. Adjuvant-induced arthritis in rats is accompanied by a loss of activity of the drug-metabolizing enzyme system and a decrease in hepatic cytochrome P-450. 2. Arthritic rats have normal serum and liver cholesterol concentrations. 3. The rate of biogenesis of cholesterol in vivo and in vitro from either [(14)C]acetate or [(14)C]mevalonate in arthritic rats was the same as or greater than that found in control rats. 4. Treatment of rats with carbon disulphide (1ml/kg) resulted in a loss of drug-metabolizing-enzyme activity and increased cholesterol biogenesis. 5. The activity of cholesterol 7alpha-hydroxylase in adjuvant-induced arthritic rats did not differ significantly from that in control rats. 6. Rats fed with cholestyramine had an elevated hepatic cholesterol 7alpha-hydroxylase activity, but neither the concentration of cytochrome P-450 nor the activity of the drug-hydroxylating enzyme, aminopyrine demethylase, was affected. 7. The relationships between drug hydroxylation and cholesterol metabolism are discussed.     

Occurrence of a new enzyme catalyzing the direct conversion of NG,NG-dimethyl-L-arginine to L-citrulline in rats

An enzyme concerned with the degradation of NG,NG-dimethyl-L-arginine to L-citrulline was investigated in rats. The enzyme purified from rat kidney catalyzed the direct conversion of NG,NG-dimethyl-L-arginine to L-citrulline with liberation of dimethylamine from the methylated guanidino moiety. The reaction required no co-factor and the maximum activity was obtained at pH 6.5. The enzyme was highly specific for NG,NG-dimethyl-L-arginine.     

Nitrogen metabolism in the facultative methylotroph Arthrobacter P1 grown with various amines or ammonia as nitrogen sources

The metabolism of trimethylamine (TMA) and dimethylamine (DMA) in Arthrobacter P1 involved the enzymes TMA monooxygenase and trimethylamine-N-oxide (TMA-NO) demethylase, and DMA monooxygenase, respectively. The methylamine and formaldehyde produced were further metabolized via a primary amine oxidase and the ribulose monophosphate (RuMP) cycle. The amine oxidase showed activity with various aliphatic primary amines and benzylamine. The organism was able to use methylamine, ethylamine and propylamine as carbon-and nitrogen sources for growth. Butylamine and benzylamine only functioned as nitrogen sources. Growth on glucose with ethylamine, propylamine, butylamine and benzylamine resulted in accumulation of the respective aldehydes. In case of ethylamine and propylamine this was due to repression by glucose of the synthesis of the aldehyde dehydrogenase(s) required for their further metabolism. Growth on glucose/methylamine did not result in repression of the RuMP cycle enzyme hexulose-6-phosphate synthase (HPS). High levels of this enzyme were present in the cells and as a result formaldehyde did not accumulate. Ammonia assimilation in Arthrobacter P1 involved NADP-dependent glutamate dehydrogenase (GDH), NAD-dependent alanine dehydrogenase (ADH) and glutamine synthetase (GS) as key enzymes. In batch cultures both GDH and GS displayed highest levels during growth on acetate with methylamine as the nitrogen source. A further increase in the levels of GS, but not GDH, was observed under ammonia-limited growth conditions in continuous cultures with acetate or glucose as carbon sources.Abbreviations HPS hexulose-6-phosphate synthase - RuMP ribulose monophosphate - DMA dimethylamine - TMA trimethylamine - TMA-NO trimethylamine-N-oxide - ICL isocitrate lyase - GS glutamine synthetase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOGAT glutamate synthase     

Partial purification and characterization of trimethylamine-N-oxide demethylase from lizardfish kidney

Trimethylamine-N-oxide demethylase (TMAOase) from lizardfish (Saurida micropectoralis) was partially purified by acidification and diethylaminoethyl (DEAE)-cellulose chromatography. The enzyme was purified 82-fold with a yield of 65.4%. The optimum pH and temperature were 7.0 and 50 degrees C, respectively. TMAOase was stable to heat treatment up to 50 degrees C and the activation energy was calculated to be 30.5 kJ mol(-1) K(-1). Combined cofactors (FeCl(2), ascorbate and cysteine) were required for full activation. FeCl(2) exhibited a higher stimulating effect on TMAOase activity than FeCl(3). At concentration less than 2 mM, ascorbate was more stimulatory to the activity than cysteine. The activity was tolerant of NaCl concentration up to 0.5 M. The enzyme had a K(m) for TMAO of 16.2 mM and V(max) of 0.35 micromol min(-1) and was able to convert TMAO to dimethylamine (DMA) and formaldehyde. The molecular mass of enzyme was estimated to be 128 kDa based on activity staining.     

Microsomal oxidation system in the course of development and aging

No substantial differences in the oxidoreductase activity of the NADH-driven electron transfer chain components in the course of development and ageing of Wistar rats aged 1, 3, 12 and 24 months were found. The content or activity of separate links of the NADPH-driven chain reached their maximal values at different age periods, being decreased with ageing. The demethylase and hydroxylase activities remained unchanged upon ageing. The ratio of the NADPH-cytochrome c reductase activity to cytochrome P-450 content was decreased in 1-, 3- and 12-months-old animals.     

Effect of dietary vitamin E on methyl ethyl ketone peroxide damage to microsomal cytochrome P-450 peroxidase

The effect of dietary vitamin E on in vivo and in vitro damage by methyl ethyl ketone peroxide (MEKP) to cytochrome P-450 and its associated enzymatic activity was studied. In vivo, MEKP damaged microsomal cytochrome P-450 and cytochrome P-450-mediated peroxidases in vitamin E-deficient rat liver. Dietary vitamin E treatment of rats protected the microsomal enzymes from peroxide damage. In vitro, the extent of MEKP inhibition was different for tetramethylphenylenediamine (TMPD)-peroxidase, NADH-peroxidase, and aminopyrine demethylase. In vitro addition of MEKP induced production of more thiobarbituric acid reacting substances (TBARS) in liver microsomes from vitamin E-deficient rats than from vitamin E-supplemented rats. When NADH and/or NADPH were supplied as reductants of MEKP, the inhibition of aminopyrine demethylase activity and the generation of TBARS by added MEKP were markedly reduced. In vivo, adequate levels of vitamin E and of NADH and NADPH are probably necessary to provide important protection to the endoplasmic reticulum during metabolism of toxic organic peroxides, such as MEKP.