Methionine Metabolites Research Articles

Sulfur-Containing Amino Acids as Precursors of Thiols in Anoxic Coastal Sediments

Sulfur-containing amino acids were examined as precursors for thiols in anoxic coastal sediments. Substrates (10 to 100 muM) were anaerobically incubated with sediment slurries; thiols were assayed as isoindole derivatives by high-performance liquid chromatography; and microbial transformations of thiols, in contrast to their chemical binding by sediment particles, were identified by inhibition with a mixture of chloramphenicol and tetracycline. Methionine and homocysteine were transformed to methanethiol and 3-mercaptopropionate (3-MPA); methionine stimulated mainly methanethiol production, whereas homocysteine generated more 3-MPA than methanethiol. 2-Keto-4-methiolbutyrate yielded results similar to those with methionine, indicating that demethiolation yields methanethiol at the keto-acid level. Glutathione gave rise to cysteine, which was further transformed to 3-mercaptopyruvate and thence to mercaptoacetate and mercaptoethanol. Mercaptoethanol was oxidized to mercaptoacetate, which was biologically consumed. In conclusion, sulfurcontaining amino acids contribute to the range of thiols that occur in anoxic coastal sediments. New metabolic and environmental transformations were identified: the production of 3-MPA as a metabolite of methionine and the transformation of mercaptopyruvate to mercaptoethanol and mercaptoacetate.

Excess Dietary Methionine Decreases Indices of Copper Status in the Rat

Two groups (n = 5) of male weanling Wistar rats were housed individually and fed copper (Cu)-deficient (0.5 mg Cu/kg) diets either with or without methionine supplementation (18 g/kg) for 49 days. Plasma caeruloplasmin (EC 1.16.3.1) and erythrocyte superoxide dismutase (EC 1.15.1.1, CuSOD) activities were measured in blood. Tissue Cu levels and the activities of cytochrome c oxidase (EC 1.9.3.1, CCO) and CuSOD were measured in the heart and liver. Hepatic activities of the sulfhydryl-sensitive enzymes, creatine kinase (EC 2.7.3.2), fumarase (EC 4.2.1.2) glutathione S-transferase (EC 2.5.1.18) and lipoamide dehydrogenase (EC 1.6.4.3) were also measured. Apart from cardiac CCO activity all of the measured indices of Cu status were found to be significantly (p less than 0.05) decreased in the methionine supplemented rats. Although fumarase activity was significantly (p less than 0.05) decreased in the methionine-supplemented animals compared with controls, the activities of the other sulfhydryl-sensitive enzymes were not significantly decreased. These results suggest that some of the toxic effects of excess dietary methionine may be mediated through interference with copper metabolism rather than through the previously postulated inhibition of sulfhydryl-sensitive enzymes by metabolites of methionine.

Synthesis and analysis of selenomethionine metabolites

This paper describes the enzymatic synthesis of selenomethionine metabolites of the transmethylation and polyamine synthesis pathways: adenosylselenomethionine, adenosylselenohomocysteine, decarboxylated adenosylselenomethionine, and methylselenoadenosine. These compounds and the corresponding methionine metabolites were simultaneously separated by a single HPLC run. The sensitivity of the HPLC method is about 20 pmol per compound. The method may be used for direct analysis of the metabolite levels in tissues or cells treated with selenomethionine and it provides an assay method for the pulse-chase type of analysis of relative flows for both selenium- and sulfurcontaining compounds in transmethylation and polyamine pathways.

Transamination of methionine in humans.

1. This study was designed to investigate the transamination pathway of methionine in humans. 2. Evidence is provided that methanethiol and its metabolites are formed via transamination of methionine. 3. Gas-liquid chromatography was used to measure serum and urinary transamination metabolites of methionine: 2-keto-4-methylthiobutyrate, 3-methylthiopropionate and methanethiol, and the metabolites of methanethiol, dimethylsulphide, protein-S-S-CH3 (a mixed disulphide of blood proteins and methanethiol) and X-S-S-CH3 (a mixed disulphide of methanethiol and another thiol with an unknown component X). 4. Methionine and the transamination intermediates were measured in 10 normal subjects, in six normal subjects after L-methionine loading (0.1 g/kg body weight) and in a male patient with hepatic methionine adenosyltransferase (EC 2.5.1.6) deficiency. 5. In the patient with methionine adenosyltransferase deficiency, at least 20% of methionine was degraded via transamination. In normal subjects transamination of methionine did exist but was quantitatively not important in methionine catabolism, not even after methionine loading. 6. The results of this study might be of importance for future studies on the role of methanethiol in hepatic encephalopathy.

Evidence for altered methionine methyl-group utilization in the diabetic rat's brain.

The methionine (MET) derivative, S-adenosylmethionine (SAM), provides methyl-groups for methylation reactions in many neural processes. In rats made diabetic with streptozotocin (SZ), brain SAM levels were generally lower (10-20%) than in controls, with a constant decrease being observed five weeks after onset of diabetes. This decrease in SAM levels may be due to reduced precursor (MET) availability because greatly elevating plasma MET concentrations in SZ diabetic rats by dietary manipulation increased their neural SAM concentrations to be approximately or even greater than (5-20%) those of controls. In contrast, neural levels of SAM's demethylated product, S-adenosylhomocysteine (SAH), were reduced to a greater extent (17-44%) than SAM levels in all groups of SZ diabetic rats independent of their plasma MET concentrations or brain SAM levels. This indicates that the decrease in SAH levels is not simply due to substrate (SAM) restriction. These changes in MET metabolites appear to be a general effect of diabetes rather than a non-pancreatic side-effect of SZ, because genetically diabetic BB Wistar rats also exhibited reduced brain SAM (25%) and brain SAH (46%) levels. These results indicate that methyl-groups from MET are handled differently in the brain of the diabetic rat, which considering the variety and importance of neural methylation reactions, could have important consequences for the diabetic.

Methionine-induced acidosis in the weanling rat.

Whole body balances of non-metabolizable base (NB) were studied in weanling rats fed a cereal-based diet with or without added L-methionine. In response to L-methionine loading (2.5 mmol day-1) the rats exhibited a significant reduction in rates of food intake (109 vs. 160 g per 8 days) and body growth (3 vs. 52 g per 8 days); fractional oxidation of absorbed dietary amino acid sulphur increased from 0.28 to 0.64; and mean urinary sulphate excretion increased from 2.3 to 14.8 mmol per 8 days. Mean rates of renal excretion of NB and filtered titratable organic acid decreased from 20 to -11 mmol per 8 days and from 22 to 8 mmol per 8 days. Balances of calcium and NB remained at reference values despite a decrease in whole blood 'base excess' from -1.0 to -6.4 mmol l-1. The concentration of NB in plasma rose but slightly. It is concluded that L-methionine loading in the weanling rat leads to extracellular non-carbonic acidosis subject to renal compensation. This acidosis is due not to retention of H+ released by ionization of endogenous sulphuric acid but possibly to accumulation of (acid) organic metabolites of methionine which are efficiently conserved by the kidneys. The rise in sulphuric acid production leads to an adaptive decrease in fractional reabsorption of filtered sulphate. Even during inhibited growth, absorbed dietary NB is retained and deposited in the skeleton, probably as calcium carbonate.

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds.

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [3H]muscimol from specific, high-affinity sites (Kd = 3.6 nM; Bmax = 3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (Kiapp) of 5000 microM, 3750 microM, and 4800 microM, respectively; while homocysteic acid (Kiapp = 4800 microM) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-L-homocysteine, L-cysteine, the convulsant L-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibiting Kiapp values of 5750 microM, 8350 microM, 5000 microM, and 510 microM, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (Ki = 20 microM) and two-site (Kiapp = 4300 microM) competitive inhibitors of [3H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.

Vitamin B6 utilization in active and inactive young men

Two groups (22–37 yr) were studied to investigate the use of vitamin B6 with exercise. Four trained men (T) were confined and studied after they ran 10 and 5 miles/day, for two 4-wk periods. They were compared with six free-living inactive men (S) consuming the same diet that provided 4.2 mg of vitamin B6 each day. N-acetyl-L-methionine, was given (60 mg/kg) as a Vitamin B6 challenge on day 28 of each period, and 24-h urinary 4-pyridoxic acid (4-PA) and methionine metabolites were determined before and after the challenge. Basal 4-PA excretion was significantly lower (p ≤ 0.05) in T than in S. In T, postmethionine 4-PA was greater than basal, while in S, postmethionine 4-PA was lower. Compared with prechallenge, postchallenge metabolites decreased in T and increased in S. Basal 4-PA excretion provides no conclusive evidence of increased need for vitamin B6 in athletes: the postload excretion suggests that exercise may promote storage of the vitamin that is available for redistribution with increased need.

Vitamin B-6 Requirement and Oral Contraceptive Use—A Concern?

There has been increasing concern as to whether oral contraceptives (OCs) increase the requirements for certain vitamins including vitamin B-6. The over 60 research studies on this relationship have used 2 parameters to evaluate vitamin B-6 status: 1) direct measures of vitamin B-6 metabolism and 2) indirect measures such as tryptophan metabolite excretion erythrocyte aminotransferase activity and stimulation and methionine metabolite excretion. Initial studies of the effect of OCs on vitamin B-6 status concentrated on altered tryptophan metabolism but there has been a recent trend toward evaluation of other indices. If only xanthurenic acid excretion is used as a criterion of OC effects the data generally suggest that OC users require supplementary B-6. Conversely analysis of plasma pyridoxal 5-phosphate (PLP) indicates such supplementation is not necessary. This points to the need for researchers to take into account the parameters that were used in presenting their findings. The observation of abnormal tryptophan is unique in that results of other tests such as the methionine load do not indicate that OC use is associated with an abnormal vitamin B-6 status. Although there may be slightly more PLP within cells where transanimase activity is elevated indicating a slightly greater need for vitamin B-6 the body appears to deal with this need by drawing on circulating PLP levels. The plasma levels of PLP in OC users are not deficient suggesting that the nutriture of OC users is not compromised.

Trace minerals, amino acids, and plasma proteins in adult men fed wheat diets

In summary, the ingestion by adult men of an all-plant diet supplying 46 gm. protein per day, primarily as bread made from white wheat four, over a period of 74 days, resulted in positive balances for copper, iron, molybdenum, selenium, and zinc. Iron balances were significantly decreased by the isonitrogenous substitution of pinto beans or peanut butter for 20 percent of the nitrogen supplied by white bread. When pinto beans or white rice substituted for 20 percent of the nitrogen of white bread, the retention of molybdenum was lower and the retention of selenium was greater. Although the men were nitrogen balance and total plasma protein concentrations were normal, the ingestion of diets providing 46 gm. protein, from all-plant sources resulted in significantly lower plasma albumin, with corresponding elevations in alpha, beta and gamma globulins. However, the substitution of pinto beans for a portion of the white bread improved plasma albumin and alpha-globulin levels. Routine clinical data obtained on patients who previously consumed strictly vegetarian diets should include determinations of plasma albumin. The ability of the subjects to maintain nitrogen balance on protein intakes of 46 gm. per day from all-plant sources appeared to be correlated with decreases in the urinary excretion and plasma concentration of alpha-aminobutyric acid, a metabolite of methionine and threonine, suggesting conservation of methionine or re-utilization of this metabolite. The possible mechanism of this adaptation to minimal protein intake, or dietary protein from all-plant sources, is discussed.

Formation of Methionine from α-Amino-n-Butyric Acid and 5′-Methylthioadenosine in the Rat

Adult rats may utilize two metabolites of methionine for the biosynthesis of this essential amino acid. In separate experiments methionine, labeled with 14C or with 35S was observed in plasma and urine following the administration of [2-14C]-alpha-amino-n-butyric acid or [35S]-5'-methylthioadenosine by stomach tube. Although alpha-amino-n-butyric acid (ABA) or homoserine, alone or with dietary sodium sulfate, choline, and/or S-methylcysteine, was not utilized for growth, weight loss in weanling rats was decreased by dietary cysteine when fed as an additive to a basal methionine-free, cysteine-free, labile methyl-free, sulfur-free diet. Following the addition of 10 mg ABA and 28 mg 5'-methylthioadenosine/day to the basal diet, growth response was equivalent to that occurring in rats receiving 27 mg of methionine/day with the basal diet. The implications of these findings for adaptation to protein restriction and a discussion of equilibrium and steady state conditions related to the increase in methionine content in the blood are presented.

Methionine-induced Ethylene Production by Penicillium digitatum

Shake cultures, in contrast to static cultures of Penicillium digitatum grown in liquid medium, were induced by methionine to produce ethylene. The induction was concentration-dependent, and 7 mM was optimum for the methionine effect. In the presence of methionine, glucose (7 mM) enhanced ethylene production but did not itself induce ethylene production. The induction process lasted several hours, required the presence of viable mycelium, exhibited a lag period for ethylene production, and was effectively inhibited by cycloheximide and actinomycin D. Thus, the methionine-induced ethylene production appeared to involve induction of an enzyme system(s). Methionine not only induced ethylene production but was also utilized as a substrate since labeled ethylene was produced from [(14)C]methionine.Following induction by the fungus, filtrates of induced shake cultures also evolved ethylene in increasing amounts by both enzymic and monenzymic reactions. Tracer experiments indicated that the ethylene released by the filtrate was derived from a fungal metabolite of methionine and not directly from methionine.

Dosage par chromatographie gazeuse avec détection en photométrie de flamme de l'acide alpha-céto-gammam-méethylthiobutyrique urinaire dans les hyperméthioninémies

In this paper we describe the determination of urinary alpha-keto-gammamethylthiobutyric acid (ACMTB) by the use of gas chromatography of its quinoxalinol derivative and specific detection of sulphur with a flame photometric detector. Using these techniques, it was possible to study the urinary elimination of this secondary metabolite of methionine in normal children, homocystinurics and in one case of hypermethioninemia. The excretion of ACMTB is normally very weak and is strongly increased when the principal pathway of methionine is blocked.

An investigation of the effects of l-methionine and related metabolites on rat behavior

l-Methionine, l-methionine plus l-serine, l-cysteine, l-serine, and betaine were tested on a modified discriminated Sidman avoidance schedule in rats. Daily injections of 250 mg/kg of these compounds were administered for at least 28 consecutive days. Schedule performance in the rat was monitored during this period and compared to preinjection saline controls. The results indicated that only methionine had significant behavior-disrupting effects. The disruption was removed by the addition of serine, suggesting that the methionine disrupting effect may have been due to one of the metabolites of methionine—homocysteine.

A study of the behavioral disruption of mice induced by l-methionine and related compounds.

Mice housed in groups normally huddle together. The daily administration of 250 mg/kg l-methionine induced disruption of this normal grouping behavior. Several compounds related to the metabolic pathway of methionine were also tested. The data indicated that the behavioral disruption observed was more likely due to the resultant increase in the level of one of the metabolites of methionine rather than to the increase in the number of available methyl groups.

Chemotaxis and methionine metabolism in Escherichia coli.

Auxotrophic strains of Escherichia coli generally are motile and chemotactic in the absence of those amino acids needed for growth. Methionine auxotrophs, however, require low levels of methionine for chemotaxis. A prototroph, grown in the presence of methionine so as to repress methionine biosynthesis, also requires methionine for chemotaxis. In the absence of methionine the bacteria are still actively motile, and thus phenotypically resemble nonchemotactic mutants.A continuous supply of methionine is necessary for a normal chemotactic response, indicating the active product is labile. Under the conditions used to demonstrate chemotaxis, the major trichloroacetic acid (TCA) soluble metabolites of methionine are spermidine, S-adenosylmethionine (SAM), and a third, as yet unidentified, compound. Spermidine is readily taken up by the bacteria, but does not replace methionine for chemotaxis. S-adenosylmethionine cannot be tested directly, as it is not taken up by normal cells. However, it is rapidly turned over and thus meets the requirement for lability. The third acid-soluble compound appears to be relatively stable metabolically.

Adenosine Triphosphate Sulfurylase from Penicillium chrysogenum: II. PHYSICAL, KINETIC, AND REGULATORY PROPERTIES

Abstract The molecular weight of Penicillium chrysogenum ATP-sulfurylase (ATP-sulfate adenylyl transferase, EC 2.7.7.4) has been shown to be between 425,000 and 440,000. Other physical parameters determined were: s20,w = 13.0, Stokes' radius = 72 A, D20,w = 2.94 x 10-7 cm3·sec-1, and v = 0.733 cm3·g-1. The carboxymethylated enzyme breaks down to subunits having a molecular weight of approximately 56,000 in the presence of 0.1% sodium dodecyl sulfate. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) reveals that there are eight reactive sulfhydryl equivalents per 440,000 g. This, together with the molecular weight of the subunit and amino acid analysis, suggests that the enzyme is an octamer containing one free sulfhydryl and four disulfides per protomer. Kinetic studies have shown that the actual substrate for the reaction is the ATP-Mg2- complex and that free ATP is a competitive inhibitor with respect to both ATP-Mg2- and MoO42- (Ki = 0.6 to 1.25 mm). The Km for ATP-Mg2- at saturating MoO42- is 4.6 x 10-5 m. The Km for MoO42- at saturating ATP-Mg2- is 1.5 x 10-4 m. In the reverse direction, the Km values for adenosine 5'-phosphosulfate and PPi are 7.1 x 10-6 and 7.7 x 10-5 m, respectively. Initial velocity studies and isotope exchange experiments show that the mechanism of the reaction is of the sequential type. The enzyme is inhibited by adenosine 5'-phosphosulfate (Ki = 4 x 10-5 m) and by sulfide. The inhibition by sulfide is sigmoidal. Sulfide also changes the molybdate concentration dependence from a hyperbolic to a sigmoidal form. The changes in the level of ATP-sulfurylase when mycelia are grown on different sulfur sources suggest that the synthesis of the enzyme is repressed by methionine, or some close metabolite of methionine.

Regulation of methionine biosynthesis in Escherichia coli: mapping of the metJ locus and properties of a metJ plus-metJ minus diploid.

MetJ(-) mutants of Escherichia coli have elevated nonrepressible levels of the enzymes of methionine biosynthesis and S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6). In E. coli, as in Salmonella typhimurium, the metJ locus is close to metB (95% cotransduction of metB and metJ markers), but in E. coli the order is reversed, with metJ mapping clockwise to metB. A stable merodiploid, heterozygous for metJ, is subject to repression by methionine. Thus, metJ functions via a diffusible product. MetJ could either be a regulatory locus or could code for an enzyme required for the synthesis of a methionine metabolite that functions in the control system.

Induction of morphogenesis by methionine starvation in Myxococcus xanthus: polyamine control.

The induction of mycrocyst formation by methionine starvation was demonstrated in Myxococcus xanthus by several methods. Growing in a defined medium (M(1)), M. xanthus had a doubling time of 6.5 hr. Four amino acids-leucine, isoleucine, valine, and glycine-were required for growth under these conditions. When the concentration of several amino acids in the medium was reduced (M(2)), the doubling time increased to 10 to 12 hr, and a requirement for methionine was observed. Methionine starvation led to a slow conversion of the population to microcysts. Under conditions of methionine prototrophy (M(1)), microcyst formation could still be triggered in exponentially growing cells by the addition of either 5 mm ethionine or 0.1 m isoleucine plus 0.1 m threonine, feedback inhibitors of methionine biosynthesis. Vegetative growth in the absence of methionine was obtained in medium M(2) if the leucine concentration was raised to its level in medium M(1). Thus, methionine biosynthesis is controlled by the exogenous concentration of the required amino acid, leucine. During an examination of the effects of methionine metabolites on microcyst formation, the involvement of polyamines in morphogenesis was uncovered. Putrescine (0.05 m) induced the formation of microcysts; spermidine (2 to 5 mm) inhibited induction by methionine starvation, ethionine, or high isoleucine-threonine. Spermidine was the only polyamine detected in M. xanthus (16.0 mug/10(9) cells). Its concentration decreased by more than 50% shortly after microcyst induction by high isoleucine-threonine. It is postulated that spermidine is an inhibitor of microcyst induction; when spermidine formation is blocked by methionine starvation, morphogenesis is induced.

Effect of Vitamin B6 Depletion in Adult Man on the Excretion of Cystathionine and Other Methionine Metabolites

The effect of vitamin B6 depletion on the metabolism of methionine in six male subjects was studied during a 49-day experiment. When subjects were fed the experimental diet, which contributed 150 g protein and 0.16 mg vitamin B6 daily, supplemented with 2.00 mg pyridoxine, the average 24-hour pre- and postmethionineloading excretion of cystathionine in the urine was 128 and 163 μmoles, respectively; about 100 μmoles cysteine sulfinic acid were excreted, but no homocystine was found. When the pyridoxine supplement was withheld, within 7 days subjects developed abnormal methionine metabolism as evidenced by an increased excretion of cystathionine following methionine loading. After 3 weeks of vitamin B6 depletion, urinary cystathionine was markedly increased, pre- and postmethionine values being 1,508 and 3,719 μmoles/24 hours; the respective values for L-cysteine sulfinic acid were 117 and 153 μmoles; the postmethionine value for homocystine was 66 μmoles and trace amounts were detected in the premethionine urines. Repletion with 2.00 mg pyridoxine daily for 2 days caused a sharp decrease in cystathionine excretion, and a 2.00 mg supplement daily for 1 week caused urinary cystathionine, L-cysteine sulfinic acid and homocystine to approach predepletion values. Urinary methionine and taurine were not affected by vitamin B6 depletion; during the repletion period, however, taurine excretion was significantly less than it was during the predepletion period.