High Concentrations Of Methionine Research Articles

Regulation of cobalamin and folate metabolism by methionine in human bone marrow cultures.

In cobalamin deficiency folate metabolism is disturbed. In the liver this deranged metabolism can be overcome by methionine, however, methionine failed to overcome this abnormality in bone marrow cultures from cobalamin deficient patients. In cobalamin deficient E. coli mutant bacteria, methionine under different conditions could either inhibit or potentiate the growth of the organism. This study was therefore initiated to test the effect of methionine, under different conditions, on bone marrow cultures. The defective DNA synthesis in megaloblastic bone marrow due to cobalamin deficiency could be corrected by the in vitro addition of low 0.27 mumol (40 micrograms) but not high 6.7 mumol (1 mg) amounts of methionine. This was measured by the ability of deoxyuridine to suppress the 3H-thymidine incorporation into DNA. The effect of methionine in facilitating de novo DNA synthesis is probably due to the catalytic action of SAM which activates cobalamin dependent methyltransferase enzyme thus potentiating the effect of cobalamin. In contrast high concentrations of methionine may inhibit this enzyme.

Distribution of seed protein fractions and amino acids in different anatomical parts of chickpea (Cicer arietinum L.) and pigeonpea (Cajanus cajan L.)

Studies on protein fractionation in seed coat, embryo, cotyledons and whole seed were made to observe the differences, if any, between chickpea and pigeonpea. Results indicated that globulin was the major fraction of embryo and cotyledons of these legumes. Seed-coat nitrogen was observed to be mostly comprised of nonprotein nitrogen and glutelin fractions and thus differed from other components in both chickpea and pigeonpea. The albumin fraction of cotyledons of both crops had the highest concentration of sulphur amino acids, methionine and cystine. Glutelin contained a considerably higher concentration of methionine and cystine than did globulin in chickpea and pigeonpea. This suggests that lines with higher glutelin should be identified to improve their protein quality. The amino acid compositions of different seed components did not show large differences between these two pulse crops.

Nutritional value of different dietary proteins to juvenile lobsters, Homarus americanus

A comparative study of the nutritional value of four proteins (casein, egg, feather and shrimp) was conducted using test diets for juvenile lobsters. Growth and survival patterns were similar for each of two groups of lobsters held at 15° C and 20° C. In general, the shrimp protein diet produced significantly better lobster growth and survival. While the feather based diet also resulted in relatively good survival, it caused the poorest growth. Amino acid analysis revealed that the shrimp protein contained higher levels of histidine than did the egg or feather proteins and higher concentrations of methionine than did the feather protein. The superiority of the shrimp-based diet, especially in relation to the casein diet, may be a result of factors other than the amino acid composition of the protein.

S-Adenosylmethionine and S-adenosylhomocystein metabolism in isolated rat liver. Effects of L-methionine, L-homocystein, and adenosine.

The effects of varying concentrations of L-methionine, L-homocysteine, and adenosine on the tissue levels of S-adenosylmethionine (AdoMet) and S-adenosyl-homocystein (AdoHcy) were investigated in perfused liver. In the normal liver, the intracellular concentration of AdoMet was dependent upon the availability of methionine. In the presence of high concentrations of methionine the maximum level of AdoMet attainable was 300 nmol/g of liver. The exogenous concentration of methionine did not alter the hepatic concentration of AdoHcy (8 to 20 nmol/g) while adenosine or homocysteine blocked hydrolysis of AdoHcy resulting in elevated levels of AdoHcy (400 to 600 nmol/g) and AdoMet (300 to 600 nmol/g). The addition of both adenosine (4mM) and homocysteine (3.4 mM) to the perfusate further increased the levels of AdoHcy (4 mumol/g) and AdoMet (1.2 mumol/g). As the concentration of AdoHcy increased, significant amounts of this compound were released into the perfusate, while AdoMet was not detected. Under all conditions where AdoHcy accumulated in the cell, a concomitant increase in the AdoMet level occurred. Apparently AdoHcy acts as a positive effector of the S-adenosylmethionine synthase. The hepatocytes did not take up significant amounts of [methyl-14C]AdoMet from the perfusate nor were any [14C]methyl groups from this compound incorporated into histones, DNA, or phospholipids. In contrast, [14C]methyl groups were readily incorporated into these macromolecules from exogenous [methyl-14C]methionine. The addition of adenosine (4 mM) and homocystein (3.4 mM) shifted the AdoMet:AdoHcy ratio from 8.2 to 0.3. Under these conditions, transmethylation was inhibited markedly.

1-Aminocyclopropane-l-carboxylic Acid - An Intermediate of the Ethylene Biosynthesis in Higher Plants

Ethylene biosynthesis in plant tissues is enhanced by addition of 1-aminocyclopropane-l-carboxylic acid (ACPCA) up to 100 times depending on the concentration. Like the endogenous ethylene biosynthesis ACPCA-dependent ethylene production is inhibited completely under anaerobic conditions. Structural integrity of the plant tissue is essential for the endogenous as well as the ACPCA-dependent ethylene biosynthesis; osmotic shock inhibits both of them. C0>Cl2, reported to inhibit ethylene biosynthesis, also inhibits ACPCA-dependent ethylene production. On the contrary, L-2-amino-4-(2-aminoethoxy)-trans-3butenoic acid (RO 4468), a potent inhibitor of the conversion of methionine to ethylene and 5-methylthioribose with S-adenosylmethionine as an intermediate, does not inhibit the ACPCA-dependent ethylene biosynthesis. High concentrations of methionine slightly stimulate ethylene production in our system but the rate of ACPCA-dependent ethylene biosynthesis is strongly inhibited. We interprete the data as follows: methionine is converted to S-adenosylmethionine as known from the literature. S-adenosylmethionine decomposes to 5'-methylthioadenosine and ACPCA. As a key intermediate the Schiff-base of ACPCA and pyridoxalphosphate, activated for example by coenzyme A as a leaving group, is suggested. Possibly already S-adenosylmethionine is converted to the CoA-activated Schiff-base of pyridoxalphosphate with the implication that the formation of free ACPCA in vivo is only a side chain reaction. Upon hydride ion uptake, the activated ACPCA decomposes to ethylene and glycine or glyoxylic acid as end products. A detailed reaction scheme is proposed.

Methionine metabolism in BHK cells: preliminary characterization of the physiological effects of cycloleucine, an inhibitor of s-adenosylmethionine biosynthesis.

Cycloleucine is in vitro a competitive inhibitor of methionine adenosyltransferase, an enzyme involved in S-adenosylmethionine biosynthesis. The physiological effects of this drug on baby hamster kidney cells have been studied. When cells are grown in a medium containing 10 micron methionine, cycloleucine is an inhibitor of cell proliferation; high concentrations of methionine are able to withdraw this inhibition suggesting that cycloleucine toxicity is related to methionine metabolism. The drug does not primarily affect methionine uptake and its subsequent use for protein biosynthesis. Cycloleucine toxicity is correlated with a block of SAM biosynthesis and nucleic acids methylations. The actions of cycloleucine on progression in the cell cycle and DNA, RNA and protein biosynthesis are studied. The implications of these results are discussed.

Altered regulation of macromolecular synthesis in methionine-inhibited cultures of Pseudomonas fluorescens UK1

Effects of high methionine concentrations on growth of Pseudomonas fluorescens UK1 are reported. The following phenomena were observed: ( i) Immediate inhibition of growth for a period corresponding to approximately half a generation. Steady-state conditions of growth were no more attained. ( ii) In spite of stringency of the macromolecular synthesis in this organism, simultaneously with the growth inhibition, the rate of labelled leucine incorporation into trichloroacetic acid (TCA)-insoluble material was reduced 60% while the rate of labelled uracil incorporation remained constant. ( iii) The organism began to liberate methanethiol half a generation after the methionine supplement. Demethiolating activity increased linearly with the cell mass. It is concluded that the inhibition of growth is not due to the liberation of methanethiol from methionine but the amino acid is able to uncouple the mechanism coordinating protein and RNA synthesis in P. fluorescens UK1.

BIOCHEMTCAL CHANGES IN THE BRAIN OF EXPERIMENTAL ANIMALS IN RESPONSE TO ELEVATED PLASMA HOMOCYSTINE AND METHIONINE

AbstractThe effects of high plasma concentrations of homocystine and methionine on the free amino acids of brain have been examined. Incorporation of the label from [35S]methionine into the free amino acid pools of rabbit brain was enhanced in response to high plasma homocystine or high plasma homocystine and mcthionine. Under comparable conditions a marked decrease in the incorporation of the label from [14C]glycine into the free pool was observed. The corresponding incorporation of 35S and 14C into brain proteins parallelled the results obtained with incorporation into the free pools of amino acids. Amino acid analyses of the free amino acid pools of rabbit brain revealed a general decrease in the concentration of amino acids in response to high plasma homocystine or high plasma homocystine and methionine. Inhibition of protein synthesis which occurs under the above experimental conditions is a general phenomenon. myelin and other brain fractions being equally affected. The decrease in concentration of brain amino acids also results in a diminution in concentration of the neurotransmitters GABA, dopamine and noradrenaline. The possible relationship of the observed changes to homocystinuria is discussed.

Methionine accumulation in villi isolated from maturing rat intestine.

1. This study compares the absorption rates of methionine in 15-day-old rats (preweaned) and 30-day-old rats (post-weaned), and the effect of varying initial substrate concentration on the absorption rate of methionine in tissue removed from specific sites in the small intestine.2. The experiments utilize a new in vitro method, the villus technique, permitting evaluation of the mucosal (villus) absorptive cells separated from the relatively non-transporting muscle and connective tissue components (non-villus tissue).3. When the villus technique was used in a comparative age study of absorption by jejunal and duodenal tissue, it was found that at initial methionine concentrations below 1.0 mM, no difference in the transport rate between preweaned and post-weaned animals was observed.4. However, in preweaned animals, methionine transport is faster in the ileum than in the jejunum or duodenum.5. With initial methionine concentration greater than 1.0 mM, duodenal and jejunal villi from preweaned animals transport methionine better than villi from older animals.6. At these higher methionine concentrations there is a slightly better transport by ileal villi from 30-day-old rats than in the younger group.7. With increasing initial concentration of methionine, physiological changes are shown to occur during maturation because transport rates into villi of mature duodenal and jejunal tissues are slower than into similar tissue from preweaned rats.8. The underlying physiological changes responsible for the transport rate differences are not known.9. The results reported indicate that following maturation, the absorbing mucosal cells from the duodenum, jejunum and ileum have an equal capacity for methionine accumulation.10. The apparent localization of the major amino acid transport to the ileum as reported by others may be an artifact reflecting the inclusion of an undetermined amount of non-absorbing muscle mass in the calculation of intestinal transport.

Effects of methionine and vitamin B 12 on the activities of methionine biosynthetic enzymes in metJ mutants of Escherichia coli K12

The effects of supplementation of growth medium with high concentrations of methionine (5 m m) and/or vitamin B 12 (10 n m) on the activities of five enzymes of the methionine regulon were measured in wild-type Escherichia coli K12, a metJ prototrophic and three metJ methionine auxotrophic derivatives. Growth on vitamin B 12 causes lowering of the activities of the non-B 12 methyltransferase while growth on methionine causes elevation of its activity in all four metJ mutants. The previous observation that this enzyme is not repressed by vitamin B 12 addition in metH mutants together with our observation that vitamin B 12 causes repression in mutants ( metF) unable to synthesize the donor for homocysteine methylation supports the model of Kung et al. (10) that the holo-B 12-methyltransferase functions as a repressor of synthesis of the non-B 12-methyltransferase. Growth on methionine causes lowering of cystathionase activity, and growth on vitamin B 12 results in elevation of cystathionase activity in a metJ prototroph and one metJ auxotroph. The metJ metA strain (RG326) has a higher than normal level of cystathionase while the metJ metF strain (RG191) has lower than normal cystathionase activity. These results indicate the existence of a metJ independent system that modulates the activity of cystathionase possibly in response to changes in concentration of unidentified metabolite(s).

Purification of methionyl-tRNA synthetase from Escherichia coli K12 by affinity chromatography.

An affinity column for the purification of methionyl‐tRNA synthetase has been prepared. Cyanogen‐bromide‐activated Sepharose 4B was reacted successively with hexamethylene‐diamine and N‐nitrophenylsulphenylmethionine succinimide ester. The nitrophenylsulphenyl protecting group of the polymer was removed under mild conditions with aqueous sodium thiosulfate. The affinity columns were equilibrated in 200 mM potassium phosphate buffer; this ionic strength proved to be sufficiently high to avoid the retention of methionyl‐tRNA synthetase by ion exchange.On columns with high methionine concentration (8–20 μmol/ml gel bed), the enzyme was strongly retained but could be selectively eluted by addition of 20 mM methionine to the buffer. However this resulted in excessive dilution of the enzyme. Highly purified enzyme could be obtained in high yield by lowering the methionine substitution of the polymer to 4.5 μmol/ml gel and by introducing the affinity chromatography after the first DEAE‐Sephadex step of the conventional purification procedure. Under these conditions no retention but sufficient retardation of the enzyme on the column was observed.The possibility of generalizing the method for the purification of other proteins recognizing an amino acid with its free amino group is discussed.

Histidine Uptake in Strains of Neurospora crassa with Normal and Mutant Transport Systems

Kinetic parameters for three systems of active histidine uptake by germinated conidia of Neurospora crassa have been measured. Each system appears to follow typical Michaelis-Menten kinetics when studied separately from the other systems. Under the conditions studied, the general amino acid transport system was found to account for the major portion of histidine uptake from low concentrations. Three types of transport mutants with altered growth inhibition patterns were selected in a histidine auxotroph. Growth of one mutant, type bas(a), could be inhibited by the addition of methionine to a histidine-supplemented medium, and another type, neu(a), could be inhibited by the addition of arginine. These mutants were shown to be lacking active histidine uptake by the basic amino acid and neutral amino acid transport systems, respectively. Another type of double mutant (his-3, neu(r)) could be inhibited only by the addition of very high concentrations of methionine in the presence of arginine and histidine, and the mutation appeared to have altered the specificity of the neutral amino acid permease.

Protein and Ornithine-δ-aminotransferase Turnover in Chang's Liver Cells

The activity of ornithine-δ-aminotransferase in Chang's liver cells in suspension culture decreased an average of 35% during 24-hour incubation in medium lacking an essential amino acid. Under the same conditions the amount of protein per cell changed very little. Protein turnover did take place however in these cells incubated in nutritionally deficient media at the rate of about 1% per hour. Concentrations of cycloheximide or puromycin low enough to permit cell survival inhibited only by 20 to 25% the incorporation of 14C-leucine into protein of cells incubated in arginine-free media. These concentrations of cycloheximide or puromycin effectively checked the synthesis of ornithine-δ-aminotransferase in cells incubated in a complete medium and maximized the decrease of enzymic activity in cells in deficient media. Actinomycin D, 5-fluorouracil, 6-mercaptopurine, or 8-azaguanine at concentrations permitting cell survival inhibited the decrease of activity of ornithine-δ-aminotransferase in cells incubated in deficient media. Significant inhibitions of this decrease of activity were obtained also when high concentrations of ornithine, lysine, valine, or methionine were added to the deficient medium.

Formation of aromatic amino acid pools in Escherichia coli K-12.

Phenylalanine, tyrosine, and tryptophan were taken up into cells of Escherichia coli K-12 by a general aromatic transport system. Apparent Michaelis constants for the three amino acids were 4.7 x 10(-7), 5.7 x 10(-7), and 4.0 x 10(-7)m, respectively. High concentrations (> 0.1 mm) of histidine, leucine, methionine, alanine, cysteine, and aspartic acid also had an affinity for this system. Mutants lacking the general aromatic transport system were resistant to p-fluorophenylalanine, beta-2-thienylalanine, and 5-methyltryptophan. They mapped at a locus, aroP, between leu and pan on the chromosome, being 30% cotransducible with leu and 43% cotransducible with pan. Phenylalanine, tyrosine, and tryptophan were also transported by three specific transport systems. The apparent Michaelis constants of these systems were 2.0 x 10(-6), 2.2 x 10(-6), and 3.0 x 10(-6)m, respectively. An external energy source, such as glucose, was not required for activity of either general or specific aromatic transport systems. Azide and 2,4-dinitrophenol, however, inhibited all aromatic transport, indicating that energy production is necessary. Between 80 and 90% of the trichloroacetic acid-soluble pool formed from a particular exogenous aromatic amino acid was generated by the general aromatic transport system. This contribution was abolished when uptake was inhibited by competition by the other aromatic amino acids or by mutation in aroP. Incorporation of the former amino acid into protein was not affected by the reduction in its pool size, indicating that the general aromatic transport system is not essential for the supply of external aromatic amino acids to protein synthesis.

Nutritional induction and suppression of fruiting in Myxococcus xanthus FBa.

A defined agar medium (A agar) containing 15 amino acids in concentrations between 0.5 and 2 mm was developed for studying the fruiting cycle of Myxococcus xanthus FBa. Cells grew only vegetatively in this medium unless the initial concentration of one of nine required or stimulatory amino acids was lowered about 50-fold. In the latter circumstance, fruiting bodies developed after several days of vegetative growth. The conclusion was that fruiting occurred when any amino acid required for normal growth became limiting in the environment. High concentrations (10 mm) of phenylalanine, tryptophan, or methionine prevented fruiting without affecting growth. Mutants requiring arginine, thymidine, or adenine could not be induced to fruit by limiting their unique requirement although they responded to the same deprivations which brought about fruiting of the wild type. A histidine auxotroph formed fruiting bodies when histidine was lowered to growth-limiting concentrations, provided that the medium was supplemented with purines. A uracil auxotroph was isolated that, perhaps secondarily, had lost some of the mechanisms which control the formation of fruiting bodies; if uracil was present, it formed fruits even when no amino acid was limiting. No concentration of uracil was sufficient to prevent fruiting. Fruiting bodies were formed when mixtures of the uracil auxotroph and wild-type cells were inoculated on A agar plus uracil, even when 75% of the cells were wild type. Microcysts of both strains were present in the fruiting bodies.

Abductin: a rubber-like protein from the internal triangular hinge ligament of pecten.

The rubber-like internal triangular hinge ligament from Pecten was studied by light and electron microscopy, x-ray diffraction, and chemical analysis. The ligament is composed of an amorphous protein, abductin. In physical properties abductin is similar to elastin and resilin but distinct by amino acid analysis. It is characterized by high concentrations of glycine and methionine.

Amino acid transport by the small intestine of the rat. The transintestinal transport of tryptophan in relation to the transport of neutral and basic amino acids.

The transintestinal transport of tryptophan by everted sacs of the rat smal intestine was studied. It was shown that: 1. 1. The transport of trytophan was enhanced by methionine, leucine and sarcocine; 2. 2. Trytophan was a potent inhibitor of the transport of leucine; 3. 3. The transport of trytophan was inhibited by higher concentrations of methionine; 4. 4. Tryptophan was a potent competitive inhibitor of the transport of lysine; 5. 5. Lysine inhibited the transport of tryptophan when a part of the intestinal capacity for transport of neutral amino acids was occupied by methionine; 6. 6. The effect of increasing the initial mucosal concentrations of tryptophan or lysine, keeping the initial serosal fluids free of amino acids, was to saturate the capacity for transport of these amino acids; 7. 7. The net transport of tryptophan and lysine from the mucosal to the serosal fluids was reduced if these amino acids were initially present in the serosal fluids. It is concluded that: 8. (i) tryptophan is a substrate for the carrier of the diamino acids as well as for the carrier of the neutral amino acids; 9. (ii) The enhancement by methionine, leucine and sarcosine of the transintestinal tryptophan transport represents a counterflow effect of these amino acids on the transport of tryptophan by diamino acid carrier; 10. (iii) The reduction caused to the transport of tryptophan and lysine by their presence in the initial serosal fluids may be explained as a kinetic consequence of the transport of these amino acids by a mobile carrier.

Threonine dehydratase activity in humans lacking cystathionine synthase

Evidence has been presented that cystathionine synthase (L-serine hydro-lyase (adding L-homocysteine) E.C.4.2.1.21) of rat liver catalyzes two additional reactions: serine deamination (L-serine hydro-lyase (deaminating) E.C.4.2.1.13) (Selim and Greenberg, 1959) and threonine deamination (L-threonine hydro-lyase (deaminating) E.C.4.2.1.16) (Goldstein, et al. , 1962) . The homogeneous protein possesses all three activities (Greenberg and Nagabhushanam, 1964). In sheep liver there are clearly two enzymes which convert serine to pyruvate and ammonia: a serine dehydratase which does not deaminate threonine (Sayre and Greenberg, 1956) and a threonine dehydratase which attacks serine at a slower rate (Nishimura and Greenberg, 1961). A recently discovered human syndrome (Carson and Neill, 1962; Field, et al. , 1962; Gerritsen, et al. , 1962; Carson, et al. , 1963; Gerritsen and Waisman, 1964) is due to a lack of activity of cystathionine synthase (Mudd, et al. , 1964; Finkelstein, et al. , 1964) . The known biochemical features of the disease include homocystinuria and abnormally high concentrations of methionine and homocystine in the plasma. We have investigated threonine metabolism in such patients to determine if they have a block in the deamination of this amino acid, and to obtain additional evidence concerning the relationship of cystathionine synthase to threonine dehydratase. In this paper we report results of assays of threonine dehydratase activity in homogenates of liver specimens from four patients lacking cystathionine synthase.

An electron-microscopic study of methionine deficient Escherichia coli

The morphology of E. coli K-12 (mutant 58/161 of Reubner) grown in a simple salt and glucose solution containing various concentrations of L-methionine and also in nutrient broth has been determined by examining electron micrographs of thin sections of osmium-fixed bacteria. The morphology of the organisms grown in high concentrations of methionine and in broth was similar. Bacteria grown in methionine deficient media contained numerous round, apparently hollow vesicles of 600 A diameter in their cytoplasm. The relationship of these vesicles to previously described cytoplasmic structures has been discussed.

Amino Acid Mixtures in the Treatment of Experimental Dietary Cirrhosis in the Rat

It has been demonstrated that cirrhosis of the liver can be produced regularly in the growing rat by diets low in protein and also deficient in choline or methionine.1-5The addition of choline or methionine to these experimental diets retards the development of the disease. However, once the disease is fully established the addition of these lipotropic factors (choline or methionine) to the low-casein diet fails to bring about adequate repair and regeneration of the liver.5,6In earlier studies7it was shown that after producing cirrhosis in rats with a diet containing 4% casein, supplementation of this diet with relatively high concentrations of choline or methionine for four months induced partial clearing of fat from the cirrhotic livers and caused the death rate to decrease but failed to promote repair and regeneration of the liver. The animals remained stunted and feeble. By contrast, in rats fed a