Evidence for S-adenosylmethionine independent catabolism of methionine in the rat

Experiments conducted with rats in vivo comparing the metabolism of methionine and S-methyl-L-cysteine and in vitro comparing methionine, S-methyl-L-cysteine and S-adenosyl-L-methionine indicate that a substantial portion of the oxidative metabolism of the methionine methyl group occurs by pathways that are independent of S-adenosylmethionine formation. Inclusion of 1.2% or 2.4% of S-methyl-L-cysteine in a diet containing 3% of L-methionine depressed the conversion of the methionine methyl and carboxyl carbons to CO2 by 39% and 28%, and 52% and 33%, respectively, for the two levels of S-methyl-L-cysteine. Inclusion of 1.65% of methionine in a diet containing 2.4% of S-methyl-L-cysteine did not affect the conversion of the methyl group of S-methylcysteine to CO2, but 3% of methionine depressed the conversion of the S-methylcysteine methyl group to CO2 to 87% of control values. Greater inhibitions were seen when these substrates were compared in a liver homogenate. In a rat liver homogenate system optimized for the conversion of the methyl group of methionine to CO2, the rate of conversion of the methyl group of S-adenosyl-L-methionine to CO2 was less than 1% of that of methionine even when the concentration of S-adenosylmethionine was saturating. Addition of saturating levels of unlabeled S-adenosymethionine to the homogenate system did not effect the rate of conversion of the methionine methyl carbon to CO2. Although S-adenosylmethionine-dependent metabolism of methionine, leading to incorporation of the methyl carbon into sarcosine and serine, could be demonstrated in liver homogenates, essentially all of the CO2 produced from the methionine methyl group was derived by a pathway or pathways independent of S-adenosylmethionine formation. Formaldehyde and formate have been tentatively identified as intermediates in catabolism of the methionine methyl group by this (these) pathway(s).     

The specific features of methionine biosynthesis and metabolism in plants

Plants, unlike other higher eukaryotes, possess all the necessary enzymatic equipment for de novo synthesis of methionine, an amino acid that supports additional roles than simply serving as a building block for protein synthesis. This is because methionine is the immediate precursor of S-adenosylmethionine (AdoMet), which plays numerous roles of being the major methyl-group donor in transmethylation reactions and an intermediate in the biosynthesis of polyamines and of the phytohormone ethylene. In addition, AdoMet has regulatory function in plants behaving as an allosteric activator of threonine synthase. Among the AdoMet-dependent reactions occurring in plants, methylation of cytosine residues in DNA has raised recent interest because impediment of this function alters plant morphology and induces homeotic alterations in flower organs. Also, AdoMet metabolism seems somehow implicated in plant growth via an as yet fully understood link with plant-growth hormones such as cytokinins and auxin and in plant pathogen interactions. Because of this central role in cellular metabolism, a precise knowledge of the biosynthetic pathways that are responsible for homeostatic regulation of methionine and AdoMet in plants has practical implications, particularly in herbicide design.     

Nursing diagnoses in patients with leukemia

Valproate (VPA) has been shown to induce neural tube defects (NTDs) in humans and mice, but the mechanism of action has not been elucidated. Folate supplementation has been reported to prevent the defect. It was the aim of our experiment to reveal effects of VPA and of folate coadministration on amino acid metabolism in an NTD mouse model. After treating pregnant mice intraperitoneally with 2.1 mmol VPA/kg body weight, plasma homocysteine concentrations were found to be increased. Coadministration of 4 mg/kg folate decreased this level. Plasma methionine levels were reduced under both experimental conditions. Fifteen min after treating mice with 3 mmol VPA/kg body weight, hepatic levels of both S-adenosylmethionine (SAM) and S-adenosylhomocysteine were found to be increased by +175% and +348%, respectively; but the levels had normalized again 30 min after VPA injection. Simultaneously, plasma methionine and serine levels had decreased by -43% and -51%, respectively, while homocysteine and cysteine increased by +71% and +81%, respectively. Reduced glutathione (GSH) decreased by -45%, but total glutathione did not change. These changes were statistically significant, and they occurred dose-dependently. We proposed that VPA induces methionine deficiency inhibition of folate metabolism and homocysteine remethylation, increase in aminothiols, and suppression of the GSH system in maternal blood within 1 h after application. These changes may be responsible for the teratogenic potential of VPA. Folate may prevent NTDs by changing homocysteine catabolism.     

Dietary betaine promotes generation of hepatic S-adenosylmethionine and protects the liver from ethanol-induced fatty infiltration

Previous studies have shown that ethanol feeding to rats alters methionine metabolism by decreasing the activity of methionine synthetase. This is the enzyme that converts homocysteine in the presence of vitamin B12 and N5-methyltetrahydrofolate to methionine. The action of the ethanol results in an increase in the hepatic level of the substrate N5-methyltetrahydrofolate but as an adaptive mechanism, betaine homocysteine methyltransferase, is induced in order to maintain hepatic S-adenosylmethionine at normal levels. Continued ethanol feeding, beyond 2 months, however, produces depressed levels of hepatic S-adenosylmethionine. Because betaine homocysteine methyltransferase is induced in the livers of ethanol-fed rats, this study was conducted to determine what effect the feeding of betaine, a substrate of betaine homocysteine methyltransferase, has on methionine metabolism in control and ethanol-fed animals. Control and ethanol-fed rats were given both betaine-lacking and betaine-containing liquid diets for 4 weeks, and parameters of methionine metabolism were measured. These measurements demonstrated that betaine administration doubled the hepatic levels of S-adenosylmethionine in control animals and increased by 4-fold the levels of hepatic S-adenosylmethionine in the ethanol-fed rats. The ethanol-induced infiltration of triglycerides in the liver was also reduced by the feeding of betaine to the ethanol-fed animals. These results indicate that betaine administration has the capacity to elevate hepatic S-adenosylmethionine and to prevent the ethanol-induced fatty liver.     

Use of 13C nuclear magnetic resonance and gas chromatography to examine methionine catabolism by lactococci

Formation of methanethiol from methionine is widely believed to play a significant role in development of cheddar cheese flavor. However, the catabolism of methionine by cheese-related microorganisms has not been well characterized. Two independent methionine catabolic pathways are believed to be present in lactococci, one initiated by a lyase and the other initiated by an aminotransferase. To differentiate between these two pathways and to determine the possible distribution between the pathways, 13C nuclear magnetic resonance (NMR) performed with uniformly enriched [13C]methionine was utilized. The catabolism of methionine by whole cells and cell extracts of five strains of Lactococcus lactis was examined. Only the aminotransferase-initiated pathway was observed. The intermediate and major end products were determined to be 4-methylthio-2-oxobutyric acid and 2-hydroxyl-4-methylthiobutyric acid, respectively. Production of methanethiol was not observed in any of the 13C NMR studies. Gas chromatography was utilized to determine if the products of methionine catabolism in the aminotransferase pathway were precursors of methanethiol. The results suggest that the direct precursor of methanethiol is 4-methylthiol-2-oxobutyric acid. These results support the conclusion that an aminotransferase initiates the catabolism of methionine to methanethiol in lactococci.     

Sex allocation and population structure in malaria and related parasitic protozoa

Here we demonstrate how sex allocation theory, one of the best verified areas of metazoan evolutionary biology, can be successfully applied to microparasitic organisms, by relating parasite prevalence and sex ratio in the Haemosporina. Members of this taxon, which includes Plasmodium, are parasitic protozoa with obligate sexual cycles in which dioecious haploid gametes drawn from the peripheral blood of a vertebrate host fuse within a dipteran vector. Consequently mating takes place within a highly subdivided population, a condition known to promote local mate competition and inbreeding and hence the evolution of female-biased sex ratios. We used an epidemiological framework to investigate mating patterns and sex ratio evolution within natural populations of these parasites. This phenotypic approach compliments more conventional biochemical approaches to the population genetics of parasitic protozoa. Data are presented which support a theoretical relation between transmission-stage sex ratio and prevalence across parasite populations. These results are consistent with a large inter-population variation in genetic structure and argue against sweeping generalizations about the clonality or otherwise of populations of these parasitic protozoa.     

DNA topoisomerases: a new twist for antiparasitic chemotherapy?

The parasitic protozoa are notorious for their bizarre cellular structures and metabolic pathways, a characteristic also true for their nucleic acids. Despite these florid differences from mammalian cells, however, it has proven surprisingly difficult to devise novel chemotherapy against these pathogens. In recent years, the DNA topoisomerases from parasites have been the focus of considerable study, not only because they are intrinsically interesting, but also because they may provide a target for much-needed new antiparasitic chemotherapy.     

Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor

The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated.     

Interaction of liver methionine adenosyltransferase with hydroxyl radical

Liver methionine adenosyltransferase (MAT) plays a critical role in the metabolism of methionine converting this amino acid, in the presence of ATP, into S-adenosylmethionine. Here we report that hydrogen peroxide (H2O2), via generation of hydroxyl radical, inactivates liver MAT by reversibly and covalently oxidizing an enzyme site. In vitro studies using pure liver recombinant enzyme and mutants of MAT, where each of the 10 cysteine residues of the enzyme subunit were individually changed to serine by site-directed mutagenesis, identified cysteine 121 as the site of molecular interaction between H2O2 and liver MAT. Cysteine 121 is specific to the hepatic enzyme and is localized at a "flexible loop" over the active site cleft of MAT. In vivo studies, using wild-type Chinese hamster ovary (CHO) cells and CHO cells stably expressing liver MAT, demonstrate that the inactivation of MAT by H2O2 is specific to the hepatic enzyme, resulting from the modification of the cysteine residue 121, and that this effect is mediated by the generation of the hydroxyl radical. Our results suggest that H2O2-induced MAT inactivation might be the cause of reduced MAT activity and abnormal methionine metabolism observed in patients with alcoholic liver disease.     

Strategies towards a better understanding of antibiotic action: folate pathway inhibition in Haemophilus influenzae as an example

Two-dimensional electrophoresis was applied to the global analysis of the cellular response of Haemophilus influenzae to sulfamethoxazole and trimethoprim, both inhibitors of tetrahydrofolate synthesis. Deregulation of the synthesis rate of 118 proteins, involved in different metabolic pathways, was observed. The regulation of the genes involved in the metabolism of the amino acids methionine, threonine, serine, glycine, and aspartate was investigated in detail by analysis of protein synthesis and Northern hybridization. The results suggested that the synthesis of methionine biosynthetic enzymes in H. influenzae is regulated in a similar fashion as in Escherichia coli. A good correlation between the results obtained by Northern hybridization and quantification of protein synthesis was observed. In contrast to trimethoprim, sulfamethoxazole triggered the increased synthesis of the heat shock proteins DnaK, GroEL, and GroES.     

IgG anti-D MAbs in tests by flow cytometry

The faunistic results regarding intestinal parasitism by protozoa and helminths in 21 primate species (three Cebidae, thirteen Cercopithecidae, one Hylobatidae, one Lemuridae, three Pongidae) are reported. The primate species were housed in four separate galleries. Six faecal samples of each host species were subjected to coprological analysis. Fifteen parasite species were detected: 11 protozoa (Entamoeba coli, E. chattoni, E. hartmanni, Iodamoeba bütschlii, Endolimax nana, Giardia intestinalis, Chilomastix mesnilii, Enteromonas hominis, Trichomonas intestinalis, Balantidium coli, and Blastocystis hominis) and 4 helminths (Ancylostoma sp., Strongyloides fuelleborni, Strongyloides sp., and Trichuris trichiura). The results reveal certain parasitic similarities between host species housed in the same gallery; however, these primate species do not always carry identical parasite species.     

Trypanosomiasis: an approach to chemotherapy by the inhibition of carbohydrate catabolism

When the infected mammalian host of Trypanosoma brucei brucei is injected with a solution of the iron chelator salicyl hydroxamic acid and glycerol, the aerobic and anaerobic glucose catabolism of the parasite is blocked and the parasite is rapidly destroyed.     

Aromatic amino acid transamination and methionine recycling in trypanosomatids

Although trypanosomatids are known to rapidly transaminate exogenous aromatic amino acids in vitro and in vivo, the physiological significance of this reaction is not understood. In postmitochondrial supernatants prepared from Trypanosoma brucei brucei and Crithidia fasciculata, we have found that aromatic amino acids were the preferred amino donors for the transamination of alpha-ketomethiobutyrate to methionine. Intact C. fasciculata grown in the presence of [15N]tyrosine were found to contain detectable [15N]methionine, demonstrating that this reaction occurs in situ in viable cells. This process is the final step in the recycling of methionine from methylthioadenosine, a product of decarboxylated S-adenosylmethionine from the polyamine synthetic pathway. Mammalian liver, in contrast, preferentially used glutamine for this reaction and utilized a narrower range of amino donors than seen with the trypanosomatids. Studies with methylthioadenosine showed that this compound was readily converted to methionine, demonstrating a fully functional methionine-recycling pathway in trypanosomatids.     

Elementary particles for models of drug dependence 10th Okey Memorial Lecture presented at the Institute of Psychiatry, London on 19th March 1997

Plasmodium falciparum secretes several proteins that cause changes in the erythrocyte membrane enabling it to survive within red blood cells. Little is known of the mechanisms involved in the secretion and targeting of parasite polypeptides to the various cell compartments. The P. falciparum gene homologous to the mammalian Sec61alpha, gene, which encodes a component of the translocation pore in the endoplasmic reticulum of eukaryotic cells, was characterised to investigate the translocation process in the parasite. PfSec61 is present as a unique copy in the parasite genome and was mapped to chromosome 13. It encodes a 40 kDa polypeptide, as shown by immunoblotting and immunoprecipitation of [35S]methionine metabolically-labelled parasite extracts. The deduced amino acid sequence of PfSec61 is 87% similar to the mammalian polypeptide, and the two proteins give similar hydropathy plots. These results strongly suggest that PfSec61 has the same topological orientation and functional role as Sec61alpha. Anti-PfSec61 antibodies were used to investigate the cellular location and kinetics of expression of the polypeptide in the parasite. Immunofluorescence confocal microscopy showed that PfSec61 was located in the parasite cytoplasm, close to the nucleus, in a position consistent with its being in the endoplasmic reticulum.     

S-adenosylmethionine metabolism and DNA methylation in hydrazine-treated rats

The treatment of rats with hepatotoxic doses of hydrazine (NH2-NH2) induces the rapid formation of 7-methylguanine and O6-methylguanine in liver DNA. The methyl moiety in these reactions might be derived from the cellular S-adenosylmethionine pool because radioactivity administered to these rats as methionine rapidly appears in the DNA as methylated guanine. An increased incorporation of radioactivity into 5-methylcytosine was previously reported followed by subsequent suppression. This increased radiolabeling of 5-methylcytosine coincided with time of maximal DNA guanine methylation. To determine the nature of S-adenosylmethionine metabolism during the period of DNA methylation induced by hydrazine treatment, and to determine if the increased radiolabeling of 5-methylcytosine at this time reflected an actual increase in 5-methylcytosine synthesis, liver DNA synthesis and S-adenosylmethionine levels and turnover were assayed. Liver S-adenosylmethionine concentrations varied slightly between control rats and hydrazinetreated rats during the first five hours after hydrazine administration, and no difference was detectable in the incorporation of administered [3H]methionine into S-adenosylmethionine. Because S-adenosylmethionine specific radioactivity in hydrazine-treated rats was not different from control rats, the previously observed increased radiolabeling of 5-methylcytosine appeared to represent an actual increase in synthesis. This conclusion was supported by finding that incorporation of radioactive thymidine into DNA was also accelerated immediately following hydrazine administration, again followed by a decrease. 5-Methylcytosine sythesis, therefore, appears to follow DNA synthesis during hydrazine toxicity, and formation of 7-methylguanine and O6-methylguanine in liver DNA of hydrazine-treated rats occurs during a short period of increased DNA sythesis and 5-methylcytosine formation very early in hydrazine toxicity.     

Control of the metabolic fate of amino acids in ruminants: a review

In general, ruminants convert ingested feed protein (N) to body tissues with low efficiency (0 to 35%). Although some of this inefficiency is due to the peculiarities of ruminal action and digestion, a large proportion is associated with metabolic events in the tissues. In the fasted condition, amino acid catabolism is greater than in the maintenance-fed animal, and perhaps 40% of this loss is due to provision of carbon sources for gluconeogenesis. The contributions of other pathways to these basal losses are poorly quantified. Below maintenance intake, insulin seems to be a major determinant of the rate of protein loss, primarily through reduction of protein degradation (especially in muscle tissue) with an accompanied decrease in the rate of branched-chain amino acid (BCAA) oxidation. At intakes above maintenance, protein anabolism and amino acid catabolism are more probably regulated by the growth hormone/insulin-like growth factor I (GH/IGF-I) axis, with the major control via alterations in protein synthesis. The actions of insulin and GH/IGF-I may provide overlapping regulatory mechanisms, which would explain the biphasic alterations in protein dynamics and amino acid catabolism observed for the ruminant between the fasted and ad libitum intake conditions. The BCAA may assume a key regulatory role in integrating the metabolism of peripheral tissues with the metabolic and oxidative functions of the liver. This integration seems well-coupled in the ruminant, for which the relationship between the extent of BCAA catabolism in peripheral and hepatic metabolism remains fairly constant under a range of nutritional and physiological conditions.     

Crossing the midline: a study of four-year-old children

Selenomethionine, an amino acid occurring in proteins in place of methionine, reacts efficiently with oxidants, e.g., peroxynitrite, a biological oxidant generated in inflammatory states, to produce the oxidation product, methionine selenoxide. The present work describes the rapid and efficient reduction of the selenoxide to selenomethionine by glutathione in a stoichiometric reaction utilizing two equivalents of thiol. This nonenzymatic reaction establishes a defense line against peroxynitrite or other oxidants by selenomethionine residues in proteins, maintained by the ubiquitous thiol, glutathione.     

Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria

Methionine synthase catalyzes the remethylation of homocysteine to methionine via a reaction in which methylcobalamin serves as an intermediate methyl carrier. Over time, the cob(I)alamin cofactor of methionine synthase becomes oxidized to cob(II)alamin rendering the enzyme inactive. Regeneration of functional enzyme requires reductive methylation via a reaction in which S-adenosylmethionine is utilized as a methyl donor. Patients of the cblE complementation group of disorders of folate/cobalamin metabolism who are defective in reductive activation of methionine synthase exhibit megaloblastic anemia, developmental delay, hyperhomocysteinemia, and hypomethioninemia. Using consensus sequences to predicted binding sites for FMN, FAD, and NADPH, we have cloned a cDNA corresponding to the "methionine synthase reductase" reducing system required for maintenance of the methionine synthase in a functional state. The gene MTRR has been localized to chromosome 5p15.2-15.3. A predominant mRNA of 3.6 kb is detected by Northern blot analysis. The deduced protein is a novel member of the FNR family of electron transferases, containing 698 amino acids with a predicted molecular mass of 77,700. It shares 38% identity with human cytochrome P450 reductase and 43% with the C. elegans putative methionine synthase reductase. The authenticity of the cDNA sequence was confirmed by identification of mutations in cblE patients, including a 4-bp frameshift in two affected siblings and a 3-bp deletion in a third patient. The cloning of the cDNA will permit the diagnostic characterization of cblE patients and investigation of the potential role of polymorphisms of this enzyme as a risk factor in hyperhomocysteinemia-linked vascular disease.     

Glycosylasparaginase-catalyzed synthesis and hydrolysis of beta-aspartyl peptides

beta-Aspartyl di- and tripeptides are common constituents of mammalian metabolism, but their formation and catabolism are not fully understood. In this study we provide evidence that glycosylasparaginase (aspartylglucosaminidase), an N-terminal nucleophile hydrolase involved in the hydrolysis of the N-glycosidic bond in glycoproteins, catalyzes the hydrolysis of beta-aspartyl peptides to form L-aspartic acid and amino acids or peptides. The enzyme also effectively catalyzes the synthesis of beta-aspartyl peptides by transferring the beta-aspartyl moiety from other beta-aspartyl peptides or beta-aspartylglycosylamine to a variety of amino acids and peptides. Furthermore, the enzyme can use L-asparagine as the beta-aspartyl donor in the formation of beta-aspartyl peptides. The data show that synthesis and degradation of beta-aspartyl peptides are new, significant functions of glycosylasparaginase and suggest that the enzyme could have an important role in the metabolism of beta-aspartyl peptides.