Lack Of Methionine Research Articles

Relative Deficiency in Albumin Methionine Content is Associated With Decreased Antioxidant Capacity of Equine Plasma

Relative to other species, horses seem particularly susceptible to oxidative stress. Plasma albumin plays an important role in preventing oxidative damage, in part due to its methionine (MET) content. Equine albumin is highly unusual in that it contains no MET residues. Whether or not this causes deficient antioxidant capacity in equine plasma relative to that of other species has not yet been explored. The objective of this study was to compare the redox status of equine (no MET) to that of bovine (moderate amount of MET) plasma. Plasma was collected from healthy, nonpregnant Quarter Horse mares (n = 10) and adult, healthy, nonpregnant, dried Holstein cows (n = 15). Measures of total antioxidant capacity and oxidative stress were assessed for each plasma sample using multiple commercially available assays: total antioxidant capacity, thiol detection, thiobarbituric acid reactive substances, and advanced oxidation protein products. Plasma from horses had significantly (P < .05) lower thiol content and thiobarbituric and reactive substances and higher advanced oxidation protein products than plasma from cattle. A difference in total antioxidant capacity was not observed; however, our study was underpowered to establish a meaningful comparison. Based on these findings, the lack of MET in equine albumin appears to translate to a lower antioxidant capacity of equine plasma. Our findings are consistent with previous reports in other species that identify MET as having an important role in the antioxidant capacity of albumin. Our results also highlight the complex system of antioxidant defenses in plasma that counteract the harmful effects of oxidants.

Activation of the antioxidant response in methionine deprived human cells results in an HSF1-independent increase in HSPA1A mRNA levels

In cells starved for leucine, lysine or glutamine heat shock factor 1 (HSF1) is inactivated and the level of the transcripts of the HSF1 target genes HSPA1A (Hsp70) and DNAJB1 (Hsp40) drops. We show here that in HEK293 cells deprived of methionine HSF1 was similarly inactivated but that the level of HSPA1A and DNAJB1 mRNA increased. This increase was also seen in cells expressing a dominant negative HSF1 mutant (HSF379 or HSF1-K80Q), confirming that the increase is HSF1 independent. The antioxidant N-acetylcysteine completely inhibited the increase in HSPA1A and DNAJB1 mRNA levels upon methionine starvation, indicating that this increase is a response to oxidative stress resulting from a lack of methionine. Cells starved for methionine contained higher levels of c-Fos and FosB mRNA, but knockdown of these transcription factors had no effect on the HSPA1A or DNAJB1 mRNA level. Knockdown of NRF2 mRNA resulted in the inhibition of the increase in the HSPA1A mRNA, but not the DNAJB1 mRNA, level in methionine starved cells. We conclude that methionine deprivation results in both the amino acid deprivation response and an antioxidant response mediated at least in part by NRF2. This antioxidant response includes an HSF1 independent increase in the levels of HSPA1A and DNAJB1 mRNA.

Crystallization and some characterization of flammulin purified from the fruit bodies of Flammulina velutipes

From the aqueous extract of fruit bodies of Flammulina velutipes, Flammulin, an antitumor substance, was purified to homogeneity judged by SDS-polyacrylamide gel electrophoresis. The purification procedure included ammonium sulfate precipitation, ethanol fractionation, DEAE-Sephadex A50 anion-exchange chromatography, crystallization and recrystallization. Flammulin was crystallized by microdialysis against 12% polyethylene glycol 6000, in 20 m m sodium phosphate buffer (pH 7.0). The crystals were rhombohedron-shaped and easy to melt when the crystallization conditions were changed. With a molecular mass of 24000, Flammulin is characterized by its lack of methionine. Amino acid composition analysis showed that the acidic amino acids were in greater number than the basic amino acids, but the isoelectric point detected by analytical isoelectric focusing was at pH 8.9.

Purification and Characterization of Fet3 Protein, a Yeast Homologue of Ceruloplasmin

The FET3 gene product of Saccharomyces cerevisiae is an essential component of the high affinity iron transport system. Based on FET3 sequence homology to the multicopper oxidase family and iron oxidation studies in spheroplasts (De Silva, D. M., Askwith, C. C., Eide, D., and Kaplan, J. (1995) J. Biol. Chem. 270, 1098-1101), it was hypothesized that the Fet3 protein (Fet3p) was a cell surface ferroxidase. To further characterize the protein, we have isolated Fet3p from yeast membranes and purified the protein to apparent homogeneity. Consistent with its localization at the plasma membrane, Fet3p is a glycosylated protein. SDS-polyacrylamide gel electrophoresis analysis showed that the protein was present in two differentially glycosylated forms of approximately 120 and 100 kDa. Purified Fet3p is a copper-containing protein that is able to catalyze the oxidation of a variety of organic compounds in addition to ferrous iron. Azide and metal chelators strongly inhibited enzyme activity. Iron appeared to be the best substrate for the enzyme, and the apparent Km for ferrous oxidation was 2 microM. Interestingly, Fet3p was able to effectively catalyze the incorporation of iron onto apotransferrin. We conclude that Fet3p is a ferro-O2-oxidoreductase in yeast, homologous to the human plasma protein ceruloplasmin.

Methionine, but Not Taurine, Protects Against Formation of Canine Pigment Gallstones

Dogs fed a marginal protein, high carbohydrate diet containing borderline amounts of methionine consistently develop pigment gallstones, marked taurine deficiency, and abnormal secretion of unconjugated bile salts. Since taurine is essential for normal secretion of bile salts in the dog, a species that cannot use glycine for bile salt conjugation, we hypothesized that taurine deficiency plays an important role in the pathogenesis of canine pigment gallstones. The rates of formation of pigment gallstones at 6 weeks were compared in dogs fed normal dog chow, lithogenic diet alone, and lithogenic diet supplemented with either taurine or methionine (45–55 mg/kg/day). No dog fed normal dog chow, but 11 of 12 dogs fed lithogenic diet, formed pigment gallstones and sludge. Supplementation of the lithogenic diet with taurine did not protect against pigment gallstones; five of six dogs developed gallstones and sludge. However, supplementation with methionine was protective, only one of six dogs was found to have a few flecks of pigmented material in its gallbladder. Interestingly, neither taurine nor methionine protected against previously observed increases in the concentrations of biliary calcium and bile salt profile indicating that at least some of the abnormalities previously observed in this model are not related to methionine/taurine deficiency. These results disprove our initial hypothesis that taurine deficiency plays an important role in the pathogenesis of pigment gallstones, but did show that a lack of methionine is linked to the formation and growth of canine pigment gallstones. The mechanism(s) leading to the formation of gallstones in dogs that are methionine deficient are is not clear from these studies.

Changes in plasma amino acid patterns and wool growth in response to abomasal injections of amino acids during late pregnancy and early lactation

Provision of sulfur-containing amino acids into the abomasum has increased wool production in non-reproducing sheep and, on the basis of such studies, they have generally been considered as the primary limiting nutrients for wool growth. The specific amino acid needs of reproducing ewes were investigated in the current study. Three groups of pregnant ewes were fitted with temporary abomasal fistulae. For the last 3-4 weeks of pregnancy and the first 3 weeks of lactation, solutions containing sodium hydroxide (as a carrier) or methionine or a mixture of valine, arginine, lysine and threonine (VALT) were injected into the abomasum twice per day. The injection of methionine did not increase protein deposition during pregnancy; this was indicated by the failure of the treatment to lower the concentration of other amino acids in plasma, or increase liveweight, lamb birth weight or wool growth. Responses in early lactation were different with the methionine injected sheep having a lower concentration of free lysine, valine, alanine and serine in plasma, indicating an increased rate of protein deposition, and a faster growth rate in the ewes. Injections of VALT caused a decrease in the concentration of cystine and methionine in plasma and depressed wool growth and tensile strength. Theresults indicate that a lack of methionine was not limiting protein deposition during late pregnancy, but may have been during early lactation. Valine, arginine, lysine and threonine were not the primary limiting amino acids in late pregnancy or early lactation.

Effects of methionine addition in soya bean or casein diets supplemented with carrageenan on weight gain and blood lipids in rats

AbstractSeven groups of 12 Wistar rats were fed during two experimental periods. During the first period of 4 weeks, rats were fed diets containing 100 g kg−1 of casein or soya‐protein. In the case of casein, diets contained casein, casein + 3.5 g kg−1 of sodium carrageenan and casein + 3.5 g kg−1 of sodium carrageenan + 1 g kg−1 of DL‐methionine. In the case of soya protein, diets contained soya protein, soya protein + 6.2 g kg−1 of sodium carrageenan, soya protein + 6.2 g kg−1 of sodium carrageenan + 1 g kg−1 of DL‐methionine and soya protein + 1 g kg−1 of DL‐methionine. During the second period of 8 weeks, all rats were fed laboratory pellets containing 170 g kg−1 of standard protein mixture. Methionine supplementation of heated soya bean meal containing carrageenan prevented the decrease of growth observed with unsupplemented diet. Methionine supplementation of casein improved protein efficiency ratio and had no effect on long‐term growth of rats because carrageenan did not cause growth retardation. At the end of second period, carrageenan ingested during the first period had no effect on plasma cholesterol. When carrageenans added to casein were ingested during the first period, plasma triglycerides were significantly lower after the second period. It would seem that carrageenans induce protein malnutrition by a lack of methionine when they are present in low protein diets with low methionine content, and that a sufficient content of methionine in diets prevents the carrageenan effect.

Acanthamoeba actin and profilin can be cross-linked between glutamic acid 364 of actin and lysine 115 of profilin.

Acanthamoeba profilin was cross-linked to actin via a zero-length isopeptide bond using carbodiimide. The covalently linked 1:1 complex was purified and treated with cyanogen bromide. This cleaves actin into small cyanogen bromide (CNBr) peptides and leaves the profilin intact owing to its lack of methionine. Profilin with one covalently attached actin CNBr peptide was purified by gel filtration followed by gel electrophoresis and electroblotting on polybase-coated glass-fiber membranes. Since the NH2 terminus of profilin is blocked, Edman degradation gave only the sequence of the conjugated actin CNBr fragment beginning with Trp-356. The profilin-actin CNBr peptide conjugate was digested further with trypsin and the cross-linked peptide identified by comparison with the tryptic peptide pattern obtained from carbodiimide-treated profilin. Amino-acid sequence analysis of the cross-linked tryptic peptides produced two residues at each cycle. Their order corresponds to actin starting at Trp-356 and profilin starting at Ala-94. From the absence of the phenylthiohydantoin-amino acid residues in specific cycles, we conclude that actin Glu-364 is linked to Lys-115 in profilin. Experiments with the isoforms of profilin I and profilin II gave identical results. The cross-linked region in profilin is homologous with sequences in the larger actin filament capping proteins fragmin and gelsolin.

Isolation and characterization of cytoplasmic fibrils from treponemes.

Electron microscopy of Triton X-100-treated whole cells of an oral treponeme, Treponema sp. strain E-21, revealed that six cytoplasmic fibrils (CFs) helically wound as a bundle in the cytoplasm. The CFs were isolated and purified by disruption and solubilization of the cells followed by CsCl density gradient centrifugation. The purified CF preparation contained mostly fibrils of about 9 nm in width and very small amounts of thinner strands of about 3 nm in diameter. The CFs were apparently seen to be a tubular structure, but the isolated CFs had narrowed sites of about 4-5 nm in width lacking lumen-like images, possibly representing twisted sites. Thus, the CF did not seem to be a tubular structure. The purified CFs were composed of one major 82 kDa protein and a few minor proteins. The CFs were destructed by treatment with proteases, 8 M urea or 4 M guanidine hydrochloride. Very low tyrosine content (0.76 mol %) and lack of methionine were characteristic features for the 82 kDa protein. The CF preparations from the other five treponemes including Treponema phagedenis and T. denticola also had 82 kDa proteins as a major component, and the 82 kDa proteins of all of the treponemes had a common antigen when examined by using antiserum against the 82 kDa protein from Treponema sp. strain E-21. Furthermore, the 82 kDa protein was demonstrated to be a principal component of the CFs of all the treponemes by immunoelectron microscopy.

THE METHYL FOLATE TRAP: A physiological response in man to prevent methyl group deficiency in kwashiorkor (methionine deficiency) and an explanation for folic-acid-induced exacerbation of subacute combined degeneration in pernicious anaemia

It is suggested that in man the methyl folate trap is a normal physiological response to impending methyl group deficiency resulting from a very low supply of methionine. This decreases cellular S-adenosyl methionine (SAM), which puts at risk important methylation reactions, including those required to maintain myelin. In order to protect these methylation reactions, the cell has evolved two mechanisms to maintain supplies of methionine and SAM as a first priority. (a) Decreased SAM causes the folate co-factors to be directed through the cycle involving 5-methyl-tetrahydrofolate (5-methyl-THF) and methionine synthetase and away from the cycles that produce purines and pyrimidines for DNA synthesis. This enhances the remethylation of homocysteine to methionine and SAM. In addition, by restricting DNA biosynthesis and with it cell division, competition for methionine for protein synthesis is reduced. Thus, whatever methionine is available is conserved for the vital methylation reactions in the nerves, brain, and elsewhere. (b) 5-methyl-THF, the form in which almost all folate is transported in human plasma, must react with intracellular homocysteine before it can be retained by the cell as a polyglutamate. Since homocysteine is derived entirely from methionine, methionine deficiency will cause intracellular folate deficiency, and the rate of mitosis of rapidly dividing cells will be reduced. Although these two processes have evolved as a response to methionine deficiency, they also occur in B 12 deficiency, which the cell mistakenly interprets as lack of methionine. The resulting response is inappropriate and gives rise to a potentially lethal anaemia. In these circumstances the methylation reactions are also partly protected by the reduced rate of cell division. This explains why administration of folic acid, which induces cell division and use of methionine in protein synthesis, impairs methylation of myelin and precipitates or exacerbates subacute combined degeneration (SCD). During folate deficiency methionine biosynthesis is also diminished. As in methionine deficiency, the body responds to decreasing availability of SAM by diverting folate away from DNA biosynthesis towards the remethylation of homocysteine to methionine and SAM. The selective use of available folate to conserve methionine, together with the ability of nerve tissue to concentrate folate from the plasma, explains the absence of SCD in folate deficiency.

Ultrapure cyanogen bromide-cleaved glucagon: Isolation in high yield by ion-exchange chromatography

Cyanogen-bromide cleaved glucagon has been extensively purified in yields of 80–85% by the use of gel filtration and by cation-exchange chromatography at pH 4.5–5.2. This pH range maintains a charge difference between the holohormone and its cleavage product, the truncated homoserine lactone derivative, yet maintains the integrity of the lactone ring. Purity is determined by the lack of methionine and the presence of homoserine following peptide hydrolysis. The homoserine lactone is opened by treatment with 0.2 n triethylamine at pH 9.5. The lactone can be reformed by treatment with trifluoroacetic acid for 1 h at room temperature although protection against photooxidation of tryptophan-25 must be provided. The homoserine lactone form binds less well to glucagon receptors than does the homoserine form. Adenylate cyclase is activated by the lactone to an extent comparable to that obtained by native hormone but at elevated concentrations. The procedures described may be useful for purification of other cyanogen bromide cleavage products and is useful for semisynthetic methods based upon cyanogen bromide-cleaved derivatives of glucagon.

Characterization of mouse tumor cell beta-lipotropin.

Mouse tumor cell beta-lipotropin (beta LPH) and gamma-lipotropin (gamma LPH) were purified from mouse pituitary tumor cell culture medium by ion exchange chromatography and gel filtration. The mouse tumor cell beta LPH was identified by immunoprecipitation with several antisera to beta-endorphin, generation of opioid bioactivity upon brief treatment with trypsin, and its identity with the molecule previously shown to serve as an intermediate in the biosynthesis of beta-endorphin. Mouse tumor cell beta LPH (Mr = 8200 +/- 250) and gamma LPH (Mr = 4600 +/- 200) are significantly smaller than known mammalian beta LPH (Mr = 10,000) and gamma LPH (Mr = 6300) molecules. The beta-endorphin region of mouse tumor cell beta LPH has the same amino acid composition as ovine, bovine, and camel beta-endorphin, and species-specific differences are thus located in the gamma LPH region of the molecule. Mouse tumor cell beta LPH and gamma LPH lack a methionine residue at what had been considered to be a highly conserved site in their beta-melanotropin-like region. A species-specific radioimmunoassay for mouse tumor cell gamma LPH was developed. Rat pituitary beta LPH and gamma LPH were shown to be similar to the corresponding mouse tumor cell molecules in size and lack of methionine in their beta-melanotropin-like segment.