N-methyltransferase Activity Research Articles

A N-methyltransferase in human brain catalyses N-methylation of 1,2,3,4-tetrahydroisoquinoline into N-methyl-1,2,3,4-tetrahydroisoquinoline, a precursor of a dopaminergic neurotoxin, N-methylisoquinolinium ion

N-Methylation of 1,2,3,4-tetrahydroisoquinoline (TIQ) present in human brain was found by a N-methyltransferase in human brain homogenate. Formation of N-methyl-1,2,3,4-tetrahydroisoquinoline (NMTIQ) from TIQ was quantitatively assayed by high-performance liquid chromatography with electrochemical detection. The reaction required S-adenosyl-L-methionine (SAM) as a methyl donor and in terms of SAM the value of the Michaelis constant, K m, and of the maximal velocity, V max, were 5.11 ± 1.69 μM and 7.31 ± 0.21 pmol/min/mg protein, respectively. The value of K m and V max in terms of TIQ were 20.9 ± 5.5 μM and 7.98 ± 1.21 pmol/min/mg protein, respectively. The optimal pH of the reaction was 8.25. A major part of the N-methyltransferase activity was found in the cytosolic fraction of human cortex. Enzymatic formation of NMTIQ indicates that in human brain this compound may be an intermediate of biosynthesis of a potent neurotoxin of dopamine metabolism, N-methylisoquinolinium ion, from naturally-occurring TIQ.

Changes in histamine metabolism in the brains of mice with streptozotocin-induced diabetes.

Histamine (HA) metabolism in the brain of mice with streptozotocin (STZ)-induced diabetes was examined. The levels of tele-methylhistamine (t-MH), a major metabolite of brain HA, significantly increased 3 and 4 weeks after STZ injection. However, the HA turnover rates in the diabetic mice, determined from the accumulation of t-MH after the administration of pargyline, were not different from the control values when the animals were allowed free access to food. When the mice were starved for 15 h 4 weeks after STZ treatment, the brain levels of L-histidine decreased significantly, whereas HA turnover increased significantly. Such changes were not observed in starved control mice. Histidine decarboxylase or HA N-methyltransferase activity did not change after starvation in either diabetic or control mice. These results show that the histaminergic (HAergic) activity in the brains of diabetic mice remains within normal range as long as the animals are allowed free access to food. However, they also indicate that a marked enhancement of HAergic activity accompanied by a decrease in the brain L-histidine level occurs in starved diabetic mice.

Histamine catabolism in proliferating skin

The histamine (HI) content and the activities of the enzymes involved in its degradation have been studied in guinea pig skin after stimulation of epidermal proliferation. As compared with unstimulated skin the HI content and histamine N-methyltransferase (HMT) activity in the stripped skin were reduced at the time of increased epidermal proliferation and were higher than the normal when the epidermis becomes hyperplastic. Diamine oxidase (DAO) followed an inverse pattern.

Comparative effects of kainic, quisqualic, and ibotenic acids on phenylethanolamine- N- methyltransferase-containing cells of rat retina

Phenylethanolamine- N-methyltransferase (PNMT) activity is located in a subpopulation of amacrine cells in the inner nuclear layer of the rat retina. Kainic, quisqualic, and ibotenic acids, all of which are analogues of glutamic acid, were injected intravitreally to the right and saline to the contralateral left eyes of adult male rats in order to determine the effect of these agents upon retinal PNMT activity. Animals were sacrificed 1 week later for tissue removal. The effect of these agents was measured by radiometric assay for PNMT. The fall in PNMT activity was used to measure the sensitivity of the PNMT-containing cells to these agents. Kainic acid was the most potent, producing the greatest reduction in PNMT activity in the smallest doses. Quisqualic acid was intermediate in potency to that of kainic and ibotenic acids. Ibotenic acid reduced PNMT activity only in extremely high doses. The PNMT-containing cells are sensitive to the toxic actions of kainic and quisqualic acids, but relatively insensitive to the actions of ibotenic acid.

S-adenosylmethionine, S-adenosylhomocysteine and adenosine system. Age-dependent availability in rat brain.

The changes in activity of N-methyltransferase 1 and [3H]-S-adenosylhomocysteine binding were determined in cortical membranes preparations from newborn and 1-, 2- and 10-month-old rats. These parameters were significantly higher: +130 and 200%, respectively, in 1-day compared to 2-month-old rats (adult). With maturation, S-adenosylmethionine levels decreased (-24%) whereas adenosine levels increased gradually from 0.17 to 6.10 nmol/mg protein in forebrain or whole brain between 1-day and 10-month-old rats. The ontogenesis of adenosine receptors was studied. The A2 receptors were not detectable at 1 day. For A1 receptors, Kd and Bmax increased with age while for A2 receptors Kd and Bmax were similar between 9 days and 2 months of age.

Putrescine and putrescine N-methyltransferase in the biosynthesis of tropane alkaloids in cultured roots of Hyoscyamus albus

The activity of arginine decarboxylase (EC 4.1.1.19) in cultured roots of Hyoscyamus albus L., which produce considerable amounts of tropane alkaloids, was twice that of ornithine decarboxylase (EC 4.1.1.17), both activities being highest during active root growth, whereas arginase (EC 3.5.3.1) activity was negligible. Actively growing roots had putrescine conjugates as their major polyamines, and spermidine was the most abundant free polyamine. Putrescine N-methyltransferase (PMT; EC 2.1.1.53) activity was high, the peak occurring on the sixth day of culture when root growth became slower. Thereafter, the free N-methylputrescine content of the roots increased and was followed by an increase in alkaloid content (mostly hyoscyamine). The amounts of arginine and, especially, of ornithine were low. No N-methylornithine was detected. The PMT activity was present only in root, shoot and cell-suspension cultures of plants that synthesized tropane alkaloids or nicotine; no enzyme activities that methylate ornithine at the δ-amino group or that decarboxylate δ-N-methylornithine were detected in any of the cultures tested. Our data indicate that tropane alkaloids in H. albus roots are synthesized by way of the symmetrical putrescine, i.e. a pathway different from that proposed by E. Leete (1962, J. Am. Chem. Soc. 84, 55) according to which these alkaloids are synthesized by way of asymmetrical δ-N-methylornithine.

Effect of vitamin B12 deficiency on S-adenosylmethionine metabolism in rats.

The effect of vitamin B12 (B12) deficiency on the levels of S-adenosylmethionine (SAM) in tissues and the activities of hepatic methionine synthase, methionine adenosyltransferase and glycine N-methyltransferase were investigated. The striking depression of methionine synthase activity was observed in all rats fed the B12-deficient diets with or without methionine supplementation for 150 days. The SAM level in liver was decreased by B12 deficiency. However, brain SAM level was not affected. The activities of hepatic methionine adenosyltransferase isozymes, alpha-form and beta-form, were decreased by B12 deficiency. Hepatic glycine N-methyltransferase activity in rats fed the low methionine-B12-deficient diet showed a tendency to lower, although the change the activity was not statistically significant, compared with B12-supplemented rats. It is proposed that the fall in the activity of hepatic methionine adenosyltransferase may be one of the causes of the decreased hepatic SAM level in B12-deficient rats.

Effects methyl-group acceptors on the regulation of plasma cholesterol level in rats fed high cholesterol diets.

The effects of methyl-group acceptors such as glycine, guanidinoacetic acid, and nicotinamide on cholesterol metabolism and phosphatidylcholine(PC) biosynthesis were investigated with rats fed a 25% casein diet containing cholesterol with or without methionine supplement. The effect of ethanolamine, an indirect methyl-group acceptor via phosphatidylethanolamine(PE) formation, was also compared with those of methyl-group acceptors. The methyl-group acceptors and ethanolamine decreased or tended to decrease plasma total cholesterol level when added to the 25% casein diet. These compounds also significantly depressed the methionine-induced enhancement of plasma cholesterol level. The activity of PE N-methyltransferase was decreased by the addition of glycine, guanidinoacetic acid, and nicotinamide, but not ethanolamine, to the reaction mixture when assayed using the postmitochondrial fraction of liver homogenate, suggesting that PE N-methyltransferase activity can be depressed by glycine N-methyltransferase, guanidinoacetic acid N-methyltransferase, and nicotinamide N-methyltransferase systems. The PE N-methyltransferase activity in liver microsomes, however, did not decrease in response to the dietary addition of methyl-group acceptors. The in vitro incorporation of [CH3-14C]methionine into PC of liver slices was also significantly inhibited by the addition of glycine and nicotinamide, but not guanidinoacetic acid and ethanolamine, to the incubation medium. It is suggested that methyl-group acceptors can decrease plasma cholesterol level at least in part through the depression of PC biosynthesis via PE N-methylation pathway, and that the mechanism for the plasma cholesterol-lowering effect of ethanolamine is different from that of methyl-group acceptors.

Phosphatidylethanolamine Levels and Regulation of Phosphatidylethanolamine N- Methyltransferase

The activity of phosphatidylethanolamine (PE) N-methyltransferase in liver microsomes, measured using endogenous microsomal PE as a substrate, was elevated 2-fold in the choline-deficient state. However, methyltransferase activity assayed in the presence of a saturating concentration of phosphatidyl-N-mono-methylethanolamine or microsomal PE was unchanged by choline deficiency. Accompanying the increase in methyltransferase activity in liver homogenates and microsomes were increased PE concentrations and an increased PE to phosphatidylcholine ratio. The concentration of other phospholipids was unchanged. Immunoblot analysis of choline-deficient and choline-supplemented rat liver microsomes using a rabbit polyclonal anti-PE N-methyltransferase antibody revealed that the amount of enzyme protein was unaltered. The regulation of methyltransferase by PE levels was also investigated in cultured hepatocytes obtained from choline-deficient rat livers. Supplementation of deficient hepatocytes with 200 microM methionine resulted in a 50% reduction in cellular PE levels over a 12-h period. PE N-methyltransferase activity assayed with endogenous PE was also reduced by 50%, but phosphatidyl-N-monomethylethanolamine-dependent activity was unchanged. A 4-h supplementation with choline did not affect PE levels or methyltransferase activity. Either methionine or choline supplementation resulted in net synthesis of cellular phosphatidylcholine. Immunoblotting of membranes from methionine-supplemented hepatocytes revealed no change in enzyme protein, a further indication that enzyme mass was constitutive, and activity was regulated by the concentration of PE.

Effect of vasopressin on the induction of adrenal tyrosine hydroxylase and phenylethanolamine N-methyltransferase

We have assessed the effect of arginine vasopressin (AVP) on adrenal tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) activities. Both enzymes show marked increases after systemic administration of AVP in the range of 66 and 100 μg/day. To determine whether the pituitary gland plays a role in these inductions, the effect of AVP (66 μg per day, given divided into 3 doses for 4 days) on the adrenal enzymes was studied in hypophysectomized rats. These animals showed induction of TH but not PNMT. This indicates that a pituitary factor(s) mediates the increase in PNMT caused by AVP. Adrenal TH activity was measured after the injection of AVP (1 or 2 μg per rat) into the lateral ventricle: there was a statistically significant increase in TH. TH was not induced in the denervated adrenal gland of rats administered AVP systemically. These findings suggest that AVP may act centrally to induce the enzyme. The continuous s.c. infusion of AVP by osmotic minipump at the rate of 1 μg/day for 6 days led to a striking increase in adrenal TH activity. However, PNMT did not increase significantly. It can be concluded that different mechanisms are involved in the induction of adrenal TH and PNMT caused by AVP. A neural mechanism is involved in TH induction, whereas PNMT induction requires release of a pituitary factor, presumably ACTH, but innervation of the adrenal is not needed for it. Moreover, the inductions of these two enzymes are differentially sensitive to the concentration of circulating AVP.

Nutritional Influences on Adrenal Chromaffin Cell Development: Comparison with Central Neurons

Neurotransmitter systems in the developing brain are generally protected from growth retardation associated with nutritional deprivation. To investigate if such protective mechanisms extend to similar tissues in the peripheral sympathetic system, maturation of the chromaffin cells of the adrenal medulla and development of their centrally derived splanchnic innervation were evaluated in rats whose nutritional status had been altered during the neonatal period by increasing (16-17 pups/litter) or decreasing (five to six pups/litter) the litter size from the standard (11-12 pups/litter). Ontogeny of adrenal catecholamine stores and activities of catecholamine-biosynthetic enzymes tyrosine hydroxylase and phenylethanolamine N-methyltransferase were monitored, along with activity of choline acetyltransferase, a marker enzyme for the preganglionic neurons innervating the chromaffin cells. Neonatal nutritional deprivation slowed body weight gain and retarded development of the chromaffin cells, as evidenced by subnormal catecholamine stores, tyrosine hydroxylase and phenylethanolamine N-methyltransferase activities. The effects persisted despite the complete recovery of body weights postweaning. The developmental alterations were not caused by overcrowding stress, as plasma corticosterone levels were not elevated in the large litter group. Neonatal nutritional enrichment promoted body weight gain but failed to enhance development of adrenal catecholamines; tyrosine hydroxylase and phenylethanolamine N-methyltransferase activities were elevated only in the preweaning period. In contrast to effects on the chromaffin cells, altered neonatal nutritional status had only minor, transient effects on the development of the centrally derived cholinergic innervation of the adrenal and produced only small changes (less than 10%) in brain tyrosine hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of phospholipid methylation in rat striatum by the corticostriatal pathway

Surgical undercutting of cortical afferents and subsequent interruption of excitatory input increased phospholipid (PL) methylation (30–40%) in rat striatal synaptosomes as early as 3 days after lesion. The increase was still present 60 days after lesion, was protein-dependent and lesion-specific. The role of the dopaminergic agents on PL methylation in decorticated rats was investigated. In contrast with sham-lesioned rats, in which dopamine stimulated PL methylation, dopamine did not further stimulate phosphatidylethanolamine N-methyltransferase (PEMT) activity in decorticated rats. In addition, dopamine antagonists reversed the increase in PL methylation in lesioned rats at concentrations that had no effect in blocking DA-stimulated PL methylation in sham-lesioned rats. The specific antagonist of N-methyl- d-aspartate receptors, 2-amino-5-phosphonovaleric acid, increased PL methylation in non-lesioned striata mimicking the effect of decortication, while N-methyl- d-aspartic acid counteracted the decortication-induced increase in PEMT activity. These data suggest a modulatory role of the cortex on PEMT activity in rat striatum.

Stimulatory effects of antiviral adenosine analogs on steroidogenesis in leydig cells

We studied the effects of 12 adenosine analogs which are active as antiviral agents on basal and LH-stimulated steroidogenesis in Leydig cells. It is shown that several of these analogs markedly stimulate the production of androgens and androgen precursors in the absence of LH. These effects are observed in interstitial cell cultures derived from immature rats as well as in freshly prepared Percoll-purified Leydig cells derived from adult mice. Some compounds (neplanocin A, S-isobutyladenosine) are active from a concentration of 10 −6 on. In the presence of maximally effective concentrations of LH or dbcAMP the stimulatory effects disappear and some compounds even become inhibitory. Only within the neplanocin series of derivatives did we observe a correlation between antiviral and steroidogenic activity. Four representative test compounds were studied in more detail: neplanocin A, 7-deazaadenosine, 4'-thioadenosine and S-isobutyladenosine. The first three significantly inhibit phospholipid N-methyltransferase activity in intact Leydig cells. However, our data do not suggest a close link between phospholipid methylation and the stimulatory or inhibitory effects of these test compounds on steroidogenesis. In cultured rat interstitial cells neplanocin A, S-isobutyladenosine and in particular 4'-thioadenosine markedly stimulate the production of cAMP. This effect is probably mediated via adenosine (A 2) receptors which are known to appear in such cultures. Comparable effects are not observed in freshly prepared mouse Leydig cells. Again, however, there is no obvious correlation between the ability of the test compounds to stimulate cAMP production and their effects on steroidogenesis. It is concluded that antiviral adenosine analogs have complex effects on Leydig cell steroidogenesis. There may not be a unifying mechanism of action underlying the various biological effects of these agonists.

Pharmacogenetics of N-methylation: heritability of human erythrocyte histamine N-methyltransferase activity.

Histamine N-methyltransferase (HNMT) catalyzes the N tau-methylation of histamine. HNMT is present in many human tissues, including the red blood cell (RBC). Our study evaluated the possible role of inheritance in the regulation of individual variations in human RBC HNMT activity. HNMT activity was measured in RBC lysates from 241 members of 51 nuclear families. After correction for the gender-specific effects of age, the frequency distribution of RBC HNMT activities was unimodal, and activities varied threefold within 2 SDs of the mean. The correlation of HNMT activities in RBCs from 45 pairs of spouses was only 0.070, indicating that shared environment did not result in similar activities among genetically unrelated individuals. Correlation coefficients were also calculated for pairs of genetically related individuals. All of these correlations were significant except the mother-oldest son correlation. The majority of the correlations did not differ significantly from those predicted for a trait with a heritability of 1.0 (100%). Our results demonstrate a significant familial aggregation of human RBC HNMT activity and suggest that inheritance may play an important role in the regulation of variation in the activity of this N-methyltransferase enzyme in the human RBC.

Structural features of potent inhibitors of rat kidney histamine N-methyltransferase

Three antimalarial drugs amodiaquine, quinacrine and chloroquine were compared side-byside with the antiseptic agent chlorhexidine and the neuromuscular blocker alcuronium for their capacity to competitively inhibit in vitro the activity of the enzyme histamine N-methyltransferase (HMT) from rat kidney over the concentration range 10 −3–10 −8. Amodiaquine was clearly the most potent HMT inhibitor followed by quinacrine, chlorhexidine, alcuronium and chloroquine. Investigation of the structure-activity relationships by examining space-filling models revealed marked similarities in the conformations of the arrangement of three N atoms in histamine and in each of the compounds tested.

Effect of chronic morphine treatment on the adrenaline biosynthesis in adrenals and brain regions of the rat

Phenylethanolamine- N-methyltransferase (PNMT) activity and tissue catecholamine content were examined after 13 days morphine treatment. Prolonged morphine treatment did not alter the PNMT activity in brain regions (A1/C1 and A2/C2 cell groups, medial basal hypothalamus, median eminence). However, the enzyme activity and the adrenaline content were increased in the adrenal medulla of male rats. In parallel, morphine treatment induced adrenal hypertrophy. In female or hypophysectomized male animals the chronic morphine treatment had no effect on adrenal weight but evoked the increase of PNMT activity. It is concluded that the morphine-induced increased adrenaline biosynthesis in the adrenal gland is not fully dependent on the intact pituitary-adrenal axis and may be mediated partly by a neural mechanism (increased splanchnic nerve activity) or by a direct effect of morphine.

Developmental Regulation of Enzymes of Indole Alkaloid Biosynthesis in Catharanthus roseus

Developing seedlings of Catharanthus roseus were analyzed for appearance of tryptophan decarboxylase (TDC), strictosidine synthase (SS), N-methyltransferase (NMT) and O-acetyltransferase (DAT) enzyme activities. SS enzyme activity appeared early after germination and was present throughout most of the developmental study. TDC activity was highly regulated and peaked over a 48 hour period achieving a maximum by day of 5 of seedling development. Both TDC and SS were present in all tissues of the seedling. NMT and DAT enzyme activities were induced after TDC and SS had peaked and these activities could only be found in hypocotyls and cotyledons. TDC, SS, and NMT did not require light for induction whereas DAT enzyme activity was increased approximately 10-fold after light treatment of dark grown seedlings.

Possible mechanism of action of SKF 64139 in vivo on rat adrenal and brain phenylethanolamine N-methyltransferase activity

SKF 64139, a specific inhibitor of the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), has been widely used as a pharmacological tool for studying the characteristics of epinephrine-containing neurons. However, the mechanism of action of this drug on PNMT in vivo has not been fully elucidated. In the present study, we traced changes of PNMT activity in rat adrenal glands and medulla oblongata between 1 and 48 hr after intraperitoneal injection of SKF 64139 (50 mg/kg body wt). Within 1 hr, enzyme activity in both tissues decreased to 10% of the respective control value. However, starting at 4 hr, activity gradually recovered from the inhibition and completely returned to the respective control level by 48 hr. Removal of the inhibitor by dialysis substantially restored the adrenal enzyme activity in 1, 2 and 4 hr groups and completely returned it to control levels in 18 and 48 hr groups. A similar pattern also seemed to hold with brain extracts. The profiles of immunotitration curves, using dialyzed tissue extracts and specific antibodies to bovine adrenal PNMT, clearly indicate that, even after dialysis, a substantial amount of inactive enzyme was present in tissue extracts from 1, 2 and 4 hr groups. In contrast, by 18 hr a very small amount of inactive enzyme was present. Throughout the experimental periods there was no noticeable differences among the control and the experimental groups in the number or intensity of immunocytochemical stained neurons with PNMT antibodies of the C 1 area of ventrolateral medulla. Judging from the data obtained by dialysis, immunochemical titration and immunocytochemical staining, recovery of PNMT activity following its inhibition by SKF 64139 was not due to irreversible inhibition of the enzyme followed by new enzyme synthesis. Instead, reversible binding of inhibitor to PNMT and its release were responsible for recovery. PNMT from the 1, 2 and 4 hr groups resisted further in vitro inhibition by SKF 64139 because the residual inhibitor was probably still bound to the enzyme.

Presence and distribution of histaminergic components in rat and bovine retina

The presence of histamine and its related enzymes, histidine decarboxylase and histamine N-methyltransferase and the subcellular distribution of the amine and of H 1-receptors were studied in the retina of two mammalian species. Histamine is present in rat and bovine retinas in concentrations (113 ± 10 and 72 ± 9 ng/g wet tissue, respectively) similar to those found in the brain. Histological examination and release experiments with Compound 48/80 performed in rat retina indicate a non mast cell location for the amine. Histidine decarboxylase and histamine N-methyltransferase activities in rat and bovine retinas were also comparable to those found in brain cortex suggesting that histamine can be synthesized and catabolyzed in situ. Subcellular fractionation of bovine retina showed that both the amine and H 1-receptors are concentrated in particulate fractions where small sized synaptosomes sediment, presumably derived from horizontal and amacrine cells. These results are in agreement with a neurotransmitter or neuromodulator role for histamine in cells of the retinal inner nuclear layer.

Correlation of sinefungin susceptibility and drug-affinity for protein carboxymethyltransferase activity in American Leishmania species

Among promastigotes of 22 different American Leishmania strains, a 5000-fold variation in sinefungin susceptibility was found, apparently independent of their taxonomic classification, although L. mexicana strains did tend to be more resistant than L. braziliensis. Protein carboxymethyltransferase (EC 2.1.1.24) and glycine N-methyltransferase (EC 2.1.1.20) activities were not substantially different in sinefungin-susceptible and -resistant American Leishmania strains. However, when [ methyl- 3H]methionine incorporation into total protein or γ-glutamyl residues of leishmanial proteins was carried out in the presence or absence of sinefungin, protein carboxymethylating activity was significantly inhibited only in sinefungin-susceptible Leishmania strains. Furthermore, when protein carboxymethyltransferase activity was purified from several leishmanial strains to a state of electrophoretic homogeneity (sp. act. = 240 nmol h −1 (mg protein) −1), the enzyme from the resistant cells showed a higher inhibition constant (mean K i 55 μM against 2 μM in susceptible cells) for sinefungin. This 28-times stronger affinity of the susceptible cell enzyme towards sinefungin despite normal protein carboxymethyltransferase specific activity seems to be a key element of the resistance mechanism of certain American Leishmania strains.