Metal chelates of 2-hydroxy-4-methylthiobutanoic acid in animal feeding. Part 2: Further characterizations, in vitro and in vivo investigations

The alpha-hydroxyacid 2-hydroxy-4-methylthiobutanoic acid (the so-called methionine hydroxy-analogue, MHA), largely used in animal nutrition as a source of methionine, forms stable metal chelates with divalent metals of formula [{CH(3)SCH(2) CH(2)CH(OH)COO}(2)M].nH(2)O. Protonation and iron(III) and copper(II) complex formation constants have been determined by potentiometry at 25 degrees C. Distribution diagrams show that no free Fe(3+) cations are present in solution at pH>2.5. ESI-MS (Electron-Spray Ionization Mass Spectrometry) investigations carried out both on iron and zinc complexes in solution have evidenced various species with different MHA/metal ratios. In vivo trials were carried out with rats. After receiving a zinc-deficient diet for 3 weeks, animals were fed the same diet added with zinc sulfate or zinc/MHA chelate; the zinc content of faeces was higher (+45%; P<0.05) in sulfate fed rats, whereas zinc retention was higher (+61%; P<0.05) in the Zn/MHA diet. Experiments in vitro with human intestinal Caco-2 cells indicated that the MHA/Fe chelate was taken up by the cells without any apparent toxic effect. The iron uptake was higher than that of iron nitrilotriacetate (Fe(3+)NTA), an effective chelate for delivering iron to milk diets. In conclusion, these data indicate that the use of MHA chelates could be a valuable tool to increase bioavailability of trace minerals and reduce the environmental impact of animal manure.     

POTENTIAL RAPID BIOASSAY FOR ALIMET® USING A METHIONINE ESCHERICHIA COLI AUXOTROPH

A potential rapid bioassay for methionine hydroxy analog (MHA) feed additive (ALIMET®) was examined using a methionine auxotroph E. coli strain. Bacterial cells were grown in minimal media containing a concentration range of 0 to 26.8 μM of either L-methionine or MHA as ALIMET®. Increasing either methionine or MHA concentration increased the growth rate of the methionine auxotroph. The estimated substrate affinities for methionine compared to MHA were not significantly different (P > 0.13) and the maximum growth rate estimates were also similar (P > 0.34). Methionine and MHA standard curves yielded linear responses (R²= 0.96) to increasing concentrations of the respective substrate. Based on these results it appears that the E. coli methionine auxotroph would have potential utility for further development of a rapid bioassay of ALIMET®.     

不同形式蛋氨酸对黄鳝生长、血清生化、血清游离氨基酸含量及肌肉品质的影响

以初始体重(30.00±0.45) g的黄鳝(Monopterus albus)为研究对象, 以低鱼粉饲料为对照组, 在低鱼粉饲料中分别添加蛋氨酸有效含量为2 g/kg的晶体蛋氨酸(C-Met)、包膜蛋氨酸(E-Met)、蛋氨酸羟基类似物钙盐(MHA-Ca)、蛋氨酸羟基类似物(MHA), 研究其在低鱼粉黄鳝饲料中的利用效果, 共5个处理组, 养殖试验持续10周。结果表明: (1)在低鱼粉饲料中添加蛋氨酸均有提高黄鳝增重率和蛋白质效率比、降低饲料系数的趋势, 其中MHA-Ca和MHA组与对照组差异显著(P<0.05)。(2)在低鱼粉饲料中添加蛋氨酸均显著提高了肠道胰蛋白酶活力、血清胆汁酸、总胆固醇、总蛋白、尿素氮和血氨含量、肌肉黏附性与胶黏性(P<0.05); 添加MHA-Ca和MHA显著提高了肠道淀粉酶活力、血清葡萄糖和高密度脂蛋白胆固醇含量、肝脏谷草转氨酶活力、全鱼粗蛋白含量、肌肉硬度、弹性和咀嚼性(P<0.05), 显著降低了黄鳝肝体比与血清谷草转氨酶活力(P<0.05)。(3)对照组在摄食6h出现蛋氨酸吸收峰值, C-Met与MHA-Ca组均在摄食后9h出现蛋氨酸吸收峰值, E-Met组在摄食后12h出现蛋氨酸吸收峰值, MHA组分别在摄食后3h和9h出现蛋氨酸峰值。结果表明, 在低鱼粉饲料中添加蛋氨酸均能提高黄鳝生长和氨基酸代谢, 且MHA-Ca与MHA效果较佳。     

Metabolism of methanol in plant cells. Carbon-13 nuclear magnetic resonance studies

Using (13)C-NMR, we demonstrate that [(13)C]methanol readily entered sycamore (Acer pseudoplatanus L.) cells to be slowly metabolized to [3-(13)C]serine, [(13)CH(3)]methionine, and [(13)CH(3)]phosphatidylcholine. We conclude that the assimilation of [(13)C]methanol occurs through the formation of (13)CH(3)H(4)Pte-glutamate (Glu)(n) and S-adenosyl-methionine, because feeding plant cells with [3-(13)CH(3)]serine, the direct precursor of (13)CH(2)H(4)Pte-Glu(n), can perfectly mimic [(13)CH(3)]methanol for folate-mediated single-carbon metabolism. On the other hand, the metabolism of [(13)C]methanol in plant cells revealed assimilation of label into a new cellular product that was identified as [(13)CH(3)]methyl-beta-D-glucopyranoside. The de novo synthesis of methyl-beta-D-glucopyranoside induced by methanol did not require the formation of (13)CH(3)H(4)Pte-Glu(n) and was very likely catalyzed by a "transglycosylation" process.     

不同形式蛋氨酸对建鲤生长性能及血清游离氨基酸含量的影响

为考察不同形式蛋氨酸对建鲤生长的作用效果, 实验以豆粕、鱼粉、棉粕为蛋白源, 配制缺乏蛋氨酸的基础饲料(对照组, 蛋氨酸含量为0.48%), 在基础饲料中分别添加晶体蛋氨酸、微囊蛋氨酸、蛋氨酸羟基类似物(MHA)及蛋氨酸羟基类似物钙盐(MHA-Ca), 使蛋氨酸含量达到0.58%, 获得5个饲料处理组, 饲养平均体重为(8.61.0) g的建鲤(Cyprinus carpio var Jian)8周。结果显示: 各组鱼体增重率分别为343.51%、350.77%、382.80%、384.02%和385.59%; 饲料系数分别为1.58、1.55、1.42、1.42和1.41; 晶体蛋氨酸组鱼体增重率、饲料系数与对照组无显著差异(P0.05), 微囊蛋氨酸组、MHA组、MHA-Ca组增重率较对照组提高11.4%、11.8%、12.2% (P0.05), 饲料系数降低10.1%、10.1%、10.8% (P0.05)。各处理组在肌肉水分、脂肪含量间无显著差异(P0.05), MHA组肌肉粗蛋白含量较晶体蛋氨酸组显著下降, 其他各组间无显著差异(P0.05)。对摄食后不同时间的血清游离氨基酸浓度变化的分析表明, 对照组在摄食后2h或3h达到峰值, 晶体蛋氨酸组、MHA组在摄食后1h达到吸收峰值, 微囊蛋氨酸组在摄食后1h或2h达到峰值, 而MHA-Ca组则在摄食后3h达到峰值。上述结果表明, 在蛋氨酸缺乏的颗粒饲料中补充晶体蛋氨酸, 对建鲤生长性能无改善作用, 而添加微囊蛋氨酸、蛋氨酸羟基类似物、蛋氨酸羟基类似物钙盐则显著提高了鱼体生长性能, 降低饲料系数。       

Biosynthesis of 3-dimethylsulfoniopropionate in Wollastonia biflora (L.) DC. Evidence that S-methylmethionine is an intermediate.

The compatible solute 3-dimethylsulfoniopropionate (DMSP) is accumulated by certain salt-tolerant flowering plants and marine algae. It is the major biogenic precursor of dimethylsulfide, an important sulfur-containing trace gas in the atmosphere. DMSP biosynthesis was investigated in Wollastonia biflora (L.) DC. [= Wedelia biflora (L.) DC., Melanthera biflora (L.) Wild, Asteraceae]. After characterizing DMSP and glycine betaine accumulation in three diverse genotypes, a glycine betaine-free genotype was chosen for radiotracer and stable isotope-labeling studies. In discs from young leaves, label from [U-14C]methionine was readily incorporated into the dimethylsulfide and acrylate moieties of DMSP. This establishes that DMSP is derived from methionine by deamination, decarboxylation, oxidation, and methylation steps, without indicating their order. Five lines of evidence indicated that methylation is the first step in the sequence, not the last. (a) In pulse-chase experiments with [14C]methionine, S-methylmethionine (SMM) had the labeling pattern expected of a pathway intermediate, whereas 3-methylthiopropionate (MTP) did not. (b) [14C]SMM was efficiently converted to DMSP but [14C]MTP was not. (c) The addition of unlabeled SMM, but not of MTP, reduced the synthesis of [14C]DMSP from [14C]methionine. (d) The dimethylsulfide group of [13CH3,C2H3]SMM was incorporated as a unit into DMSP. (e) When [C2H3,C2H3]SMM was given together with [13CH3]methionine, the main product was [C2H3,C2H3]DMSP, not [13CH3,C2H3]DMSP or [13CH3,13CH3]DMSP. The stable isotope labeling results also show that the SMM cycle does not operate at a high level in W. biflora leaves.     

Comparison of the Efficacies of Chloromethane, Methionine, and S-Adenosylmethionine as Methyl Precursors in the Biosynthesis of Veratryl Alcohol and Related Compounds in Phanerochaete chrysosporium

The effect on veratryl alcohol production of supplementing cultures of the lignin-degrading fungus Phanerochaete chrysosporium with different methyl-(sup2)H(inf3)-labelled methyl precursors has been investigated. Both chloromethane (CH(inf3)Cl) and l-methionine caused earlier initiation of veratryl alcohol biosynthesis, but S-adenosyl-l-methionine (SAM) retarded the formation of the compound. A high level of C(sup2)H(inf3) incorporation into both the 3- and 4-O-methyl groups of veratryl alcohol occurred when either l-[methyl-(sup2)H(inf3)]methionine or C(sup2)H(inf3)Cl was present, but no significant labelling was detected when S-adenosyl-l-[methyl-(sup2)H(inf3)]methionine was added. Incorporation of C(sup2)H(inf3) from C(sup2)H(inf3)Cl was strongly antagonized by the presence of unlabelled l-methionine; conversely, incorporation of C(sup2)H(inf3) from l-[methyl-(sup2)H(inf3)]methionine was reduced by CH(inf3)Cl. These results suggest that l-methionine is converted either directly or via an intermediate to CH(inf3)Cl, which is utilized as a methyl donor in veratryl alcohol biosynthesis. SAM is not an intermediate in the conversion of l-methionine to CH(inf3)Cl. In an attempt to identify the substrates for O methylation in the metabolic transformation of benzoic acid to veratryl alcohol, the relative activities of the SAM- and CH(inf3)Cl-dependent methylating systems on several possible intermediates were compared in whole mycelia by using isotopic techniques. 4-Hydroxybenzoic acid was a much better substrate for the CH(inf3)Cl-dependent methylation system than for the SAM-dependent system. The CH(inf3)Cl-dependent system also had significantly increased activities toward both isovanillic acid and vanillyl alcohol compared with the SAM-dependent system. On the basis of these results, it is proposed that the conversion of benzoic acid to veratryl alcohol involves para hydroxylation, methylation of 4-hydroxybenzoic acid, meta hydroxylation of 4-methoxybenzoic acid to form isovanillic acid, and methylation of isovanillic acid to yield veratric acid.     

Effect of methionine and its related compounds on rumen bacterial activity

The effect of several methionine sources (L‐methionine = L‐MET; DL‐ methionine = DL‐MET, DL‐S‐methyl‐methionine‐sulphonium‐chloride = SMM; N‐hydroxymethyl‐DL‐methionine‐Ca = NHM; methionine‐hydroxy‐analog free acid=MHA; methionine‐sulphoxyde=MSO) on rumen bacterial growth was studied using a new methodical approach which utilises a methionine free assay medium (Bacto Methionine Assay Media, Difco) supplemented by increasing quantities of the methionine sources and inoculated with one drop of diluted rumen bacteria. The optical density was measured after 18 h incubation on 39 °C.

L‐ and DL‐MET promoted the highest growth response, while SMM and NHM exerted significantly (p < 0.05) lower optical densities. MHA and MSO showed no growth response.

The methodical approach and the possible bacterial strains, which might have contributed to the growth response have been discussed.     

Production and fate of methylated sulfur compounds from methionine and dimethylsulfoniopropionate in anoxic salt marsh sediments

Anoxic salt marsh sediments were amended with dl-methionine and dimethylsulfoniopropionate (DMSP). Microbial metabolism of methionine yielded methane thiol (MSH) as the major volatile organosulfur product, with the formation of lesser amounts of dimethylsulfide (DMS). Biological transformation of DMSP resulted in the rapid release of DMS and only small amounts of MSH. Experiments with microbial inhibitors indicated that production of MSH from methionine was carried out by procaryotic organisms, probably sulfate-reducing bacteria. Methane-producing bacteria did not metabolize methionine. The involvement of specific groups of organisms in DMSP hydrolysis could not be determined with the inhibitors used, because DMSP was hydrolyzed in all samples except those which were autoclaved. Unamended sediment slurries, prepared from Spartina alterniflora sediments, contained significant (1 to 10 muM) concentrations of DMS. Endogenous methylated sulfur compounds and those produced from added methionine and DMSP were consumed by sediment microbes. Both sulfate-reducing and methane-producing bacteria were involved in DMS and MSH consumption. Methanogenesis was stimulated by the volatile organosulfur compounds released from methionine and DMSP. However, apparent competition for these compounds exists between methanogens and sulfate reducers. At low (1 muM) concentrations of methionine, the terminal S-methyl group was metabolized almost exclusively to CO(2) and only small amounts of CH(4). At higher (>100 muM) concentrations of methionine, the proportion of the methyl-sulfur group converted to CH(4) increased. The results of this study demonstrate that methionine and DMSP are potential precursors of methylated sulfur compounds in anoxic sediments and that the microbial community is capable of metabolizing volatile methylated sulfur compounds.     

Solution (sup13)C Nuclear Magnetic Resonance Spectroscopic Analysis of the Amino Acids of Methanosphaera stadtmanae: Biosynthesis and Origin of One-Carbon Units from Acetate and Carbon Dioxide

We found that general pathways for amino acid synthesis of Methanosphaera stadtmanae, a methanogen that forms CH(inf4) from H(inf2) and methanol, resembled those of methanogens that form CH(inf4) from CO(inf2) or from the methyl group of acetate. We determined the incorporation of (sup14)C-labeled CO(inf2), formate, methanol, methionine, serine, and acetate into cell macromolecules. Labeling of amino acid carbons was determined by solution nuclear magnetic resonance spectroscopy after growth with (sup13)C-labeled acetate, CO(inf2), serine, and methanol. The (alpha) and (beta) carbons of serine and alanine were formed from carboxyl and methyl carbons of acetate, respectively, and the amino acid carboxyl groups were formed from CO(inf2). This indicates that pyruvate was formed by reductive carboxylation of acetate. Labeling of the methyl carbon of methionine indicated that the major route of synthesis was from the hydroxymethyl carbon of serine that arises from the methyl carbon of acetate. Methanol was a minor source of the methyl of methionine. Unambiguous assignment was made of the sources of all carbons of histidine. Labeling of the histidine 7 position ((epsilon) carbon) was consistent with formation from the C-2 of the purine ring of ATP and the origin of the C-2 from a formyl unit derived from the hydroxymethyl carbon of serine.     

Sustained endothelial nitric-oxide synthase activation requires capacitative Ca2+ entry

Endothelial nitric-oxide synthase (eNOS), a Ca(2+)/calmodulin-dependent enzyme, is critical for vascular homeostasis. While eNOS is membrane-associated through its N-myristoylation, the significance of membrane association in locating eNOS near sources of Ca(2+) entry is uncertain. To assess the Ca(2+) source required for eNOS activation, chimera containing the full-length eNOS cDNA and HA-tagged aequorin sequence (EHA), and MHA (myristoylation-deficient EHA) were generated and transfected into COS-7 cells. The EHA chimera was primarily targeted to the plasma membrane while MHA was located intracellularly. Both constructs retained enzymatic eNOS activity and aequorin-mediated Ca(2+) sensitivity. The plasma membrane-associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca(2+) concentration, demonstrating preferential sensitivity to Ca(2+) originating from intracellular pools (MHA) or from capacitative Ca(2+) entry (EHA). Measurements of eNOS activation in intact cells revealed that the eNOS enzymatic activity of EHA was more sensitive to Ca(2+) influx via capacitative Ca(2+) entry than intracellular release, whereas MHA eNOS activity was more responsive to intracellular Ca(2+) release. When eNOS activation by CCE was compared with that generated by an equal rise in [Ca(2+)](i) due to the Ca(2+) ionophore ionomycin, a 10-fold greater increase in NO production was found in the former condition. These results demonstrate that EHA and MHA chimera are properly targeted and retain full functions of eNOS and aequorin, and that capacitative Ca(2+) influx is the principle stimulus for sustained activation of eNOS on the plasma membrane in intact cells.     

Naturally occurring cobalamins have antimalarial activity

The acquisition of resistance by malaria parasites towards existing antimalarials has necessitated the development of new chemotherapeutic agents. The effect of vitamin B(12) derivatives on the formation of beta-haematin (synthetic haemozoin) was determined under conditions similar to those in the parasitic food vacuole (using chloroquine, a known inhibitor of haemozoin formation for comparison). Adenosylcobalamin (Ado-cbl), methylcobalamin (CH(3)-cbl) and aquocobalamin (H(2)O-cbl) were approximately forty times more effective inhibitors of beta-haematin formation than chloroquine, cyanocobalamin (CN-cbl) was slightly more inhibitory than chloroquine, while dicyanocobinamide had no effect. It is proposed that the cobalamins exert their inhibitory effect on beta-haematin formation by pi-interactions of their corrin ring with the Fe(III)-protoporphyrin ring and by hydrogen-bonding using their 5,6-dimethylbenzimidazole/ribose/sugar side-chain. The antimalarial activity for the cobalamins (Ado-cbl>CH(3)-cbl>H(2)O-cbl>CN-cbl) was found to be less than that for chloroquine or quinine. Ado-cbl, CH(3)-cbl and CN-cbl do not accumulate in the parasite food vacuole by pH trapping, but H(2)O-cbl does. Unlike humans, the malaria parasite has only one enzyme that uses cobalamin as a cofactor, namely methionine synthase, which is important for growth and metabolism. Thus cobalamins in very small amounts are necessary for Plasmodium falciparum growth but in larger amounts they display antimalarial properties.     

Methionine oxidation of Sup35 protein induces formation of the [PSI+] prion in a yeast peroxiredoxin mutant

The frequency with which the yeast [PSI(+)] prion form of Sup35 arises de novo is controlled by a number of genetic and environmental factors. We have previously shown that in cells lacking the antioxidant peroxiredoxin proteins Tsa1 and Tsa2, the frequency of de novo formation of [PSI(+)] is greatly elevated. We show here that Tsa1/Tsa2 also function to suppress the formation of the [PIN(+)] prion form of Rnq1. However, although oxidative stress increases the de novo formation of both [PIN(+)] and [PSI(+)], it does not overcome the requirement of cells being [PIN(+)] to form the [PSI(+)] prion. We use an anti-methionine sulfoxide antibody to show that methionine oxidation is elevated in Sup35 during oxidative stress conditions. Abrogating Sup35 methionine oxidation by overexpressing methionine sulfoxide reductase (MSRA) prevents [PSI(+)] formation, indicating that Sup35 oxidation may underlie the switch from a soluble to an aggregated form of Sup35. In contrast, we were unable to detect methionine oxidation of Rnq1, and MSRA overexpression did not affect [PIN(+)] formation in a tsa1 tsa2 mutant. The molecular basis of how yeast and mammalian prions form infectious amyloid-like structures de novo is poorly understood. Our data suggest a causal link between Sup35 protein oxidation and de novo [PSI(+)] prion formation.     

Nutritive value of lupin-seed protein and leaf-protein concentrates supplemented with various sulphur sources

The effect of DL methionine, methionine hydroxyanalogue (MHA) and sodium sulphate on the nutritive value of lupin-seed protein and leaf-protein concentrate (LPC) from lucerne for rats and chicks was evaluated. Methionine and MHA markedly improved the biological value of both lupin- and LPC-proteins, but the effect of sodium sulphate was not significant.The supplement of organic sulphur-compounds improved the performance of chicks fed on lupin-seed meal, but the effect of sodium sulphate was negligible. There was no significant response to any of the organic and inorganic sulphur compounds in chicks fed on LPC diets. These results suggest that some factor other than methionine defiency is limiting the nutritive value of LPC for these animals.     

Dissociation constants of protonated methionine species in NaCl media

The sulfur-containing amino acid, methionine, has a role in the physiological environment because of its strong interactions with metals. To understand these interactions of metals with methionine, one needs reliable dissociation constants for the protonated methionine species (NH(3)(+)CH(CH(2)CH(2)SCH(3))COOH; H(2)B(+)). The values of stoichiometric dissociation constants, pK(i)*, for protonated methionine species (H(2)B(+) if H(+)+HB, K(1); HB if H(+)+B(-), K(2)) were determined from potentiometric measurements in NaCl solutions as a function of ionic strength, 0.25-6.0 mol (kg H(2)O)(-1) and temperature (5-45 degrees C). The results were extrapolated to pure water using the Pitzer equations to estimate the activity of H(+), H(2)B(+), HB and B(-) as a function of ionic strength and temperature. The resulting thermodynamic values of K(1) and K(2) were fit to the equations (T/K): ln K(1)=69.0013-3496.58/(T/K)-10.9153 ln (T/K); ln K(2)=116.4162-10638.02/(T/K)-18.0553 ln (T/K) with standard errors of 0.003 and 0.033, respectively, for ln K(1)* and ln K(2)*. Pitzer interaction parameters (lambda(HB-Na) and zeta(HB-Na-Cl)) for the neutral HB were determined from literature data. The Pitzer parameters (beta(0)(H(2)BCl), beta(1)(H(2)BCl) and C(phi)(H(2)BCl)) for the interactions of H(2)B(+) with Cl(-) and Na(+) with and B(2-) (beta(0)(NaB), beta(1)(NaB) and C(phi)(NaB)) were also determined. These coefficients can be used to make reasonable estimates of the activity coefficients of methionine species and the pK(i)(*) for the dissociation of methionine in physiological solutions, composed mostly of NaCl over a wide range of temperature and ionic strength.     

Methionine Recycling in Brain: A Role for Folates and Vitamin B-12

Abstract: The recycling of methionine via homocysteine was measured in vivo in brain. After constant intravenous infusions (5 h) of both [³H-methyl] methionine and [³⁵S]methionine into rats, the ratios of [³H-methyl]methionine to [³⁵S]methionine in liver, brain and plasma were determined, Similar experiments were performed in rabbits, except that the [³H-methyl]- and [³S]methionine were injected intraventricularly. If the methyl group of methionine was removed with the formation of homocysteine and then replaced by another (unlabeled) methyl group, the specific activity of the [³H-methyl]methionine would decrease more than that of [³⁵S]methionine; i.e., the ratio of [³H-methyl]- to [³⁵S]methionine in the tissue would decline. The results showed that the ratios of [³H-methyl]- to [³⁵S]methionine in liver and brain were less than the same ratio in plasma in the rats. The comparable ratios in the brain and CSF of rabbits were less than the ratio in the injectate. Since brain contains only one enzyme capable of remethylating homocysteine to methionine, the vitamin B-12–dependent methyltetrahydrofolate-homocysteine methyltransferase (EC 2.1.1.13), our results for methionine recycling via homocysteine in brain strongly support the activity of this enzyme in brain in vivo.     

Transsulphuration and methylation of homocysteine in control and mutant human fibroblasts

The ability of human skin-fibroblasts in monolayer culture to carry out transsulphuration and remethylation of homocysteine has been tested. The conversion of homocyst(e)ine to cyst(e)ine and methionine was studied in control and mutant cells by incubation for 16 h with l-[³⁵S]homocystine. Labelled cysteic acid and methionine sulphone were found in hydrolysates of oxidized cell proteins. The quantities found were dependent on the time of incubation and were used as a measure of cyst(e)ine and methionine formation, respectively. In control cells, labelled cyst(e)ine and labelled methionine were found. In cystathionine β-synthase-deficient cell lines, labelled cyst(e)ine formation was reduced, while labelled methionine formed was similar to that of controls, indicating the role of transsulphuration in the formation of cyst(e)ine observed in control cells. In a 5,10-methylenetetrahydrofolate reductase-deficient cell line, labelled methionine formation was reduced, indicating the role of N-5-methyltetrahydrofolate-requiring methylation of homocysteine in the formation of methionine observed in control cells.     

Biosynthesis of tissue inhibitor of metalloproteinases by human fibroblasts in culture. Stimulation by 12-O-tetradecanoylphorbol 13-acetate and interleukin 1 in parallel with collagenase

Biosynthesis of the glycoprotein tissue inhibitor of metalloproteinases (TIMP) by human fibroblasts in culture has been characterized by functional assays, immunoprecipitation, and immunocytochemistry with a monospecific antiserum. As determined by radiolabeling with [35S]methionine, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the secreted form of TIMP had an Mr of 29,000, whereas the form associated with the cell layer had an Mr of 24,000. Unstimulated human lung fibroblasts (HFL-1) secreted TIMP at the rate of approximately 2 micrograms/10(6) cells/24 h, and normal foreskin fibroblasts (HS 27) and skin fibroblasts from a patient with Hurler's disease (GM 1391) secreted TIMP at 0.3 and 0.2 micrograms/10(6) cells/24 h, respectively. Secretion of TIMP was stimulated up to 10-fold by treating the cells with 20-100 ng/ml of 12-O-tetradecanoylphorbol 13-acetate or 10 units/ml of human interleukin 1. In the stimulated HFL-1 cells, TIMP accounted for 0.03-0.09% of the total [35S]methionine incorporated into protein, and 0.3-0.8% of the [35S]methionine in secreted protein. Although TIMP accounted for a relatively small proportion of total protein synthesis of the fibroblasts, greater than 80% of untreated and greater than 95% of stimulated fibroblasts synthesized TIMP, as determined by indirect immunofluorescence. The treatments of the human fibroblasts that increased TIMP secretion also induced synthesis and secretion of proenzyme forms of collagenase, indicating that degradative enzymes and their controlling inhibitors may be synthesized in parallel under certain conditions.     

Assay and regulation of S-adenosylmethionine synthetase in Saccharomyces cerevisiae and Candida utilis.

A simple and sensitive assay for S-adenosylmethionine (SAM) synthetase is described which depends on the quantitative separation of the product, [14CH3]S-adenosylmethionine, from the substrate, L-[14CH3]methionine, on a Bio-Rex 70 column. L-Methionine protects the enzyme during preparation of cell extracts by sonic treatment but causes repression of enzyme activity during growth of Candida utilis. The presence of 5 mM methionine in the growth medium repressed SAM synthetase specific activity threefold compared to the specific acitivity of the enzyme isolated from cells grown in unsupplemented medium. Conversely, the presence of methionine in the growth medium resulted in an 80-fold increase in the intracellular concentration of SAM as compared to the Sam accumulated intracellularly in unsupplemented cultures.     

Studies on reactions of oxidizing sulfur-sulfur three-electron-bond complexes and reducing alpha-amino radicals derived from OH reaction with methionine in aqueous solution

The technique of pulse radiolysis with spectrophotometric detection has been used to investigate the possibility of electron transfer reactions between oxidizing sulfur-sulfur three-electron-bond complexes (Met2/S thereforeS+), or reducing alpha-amino radicals (CH3SCH2CH2CH.NH2) derived from reaction of methionine with OH radicals and hydroxycinnamic acid (HCA) derivatives, riboflavin (RF) or flavin adenine dinucleotide (FAD), respectively. The HCA derivatives, such as caffeic acid, ferulic acid, sinapic acid and chlorogenic acid, widely distributed phenolic acids in fruit and vegetables, have been identified as good antioxidants previously can rapidly and efficiently repair oxidizing three-electron-bond complexes via electron transfer. RF and FAD can oxidize reducing alpha-amino radicals derived from methionine. The electron transfer rate constants approximately 10(9) dm3 x mol(-1)x s(-1) were determined by following the build-up kinetics of species produced.