Determination of S-adenosylmethionine and S-adenosylhomocysteine in blood plasma by UPLC with fluorescence detection.

A validated approach to determine various methionine cycle metabolites (S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine) in human blood plasma is offered. The approach is based on solid-phase extraction (with grafted phenylboronic acid) and derivatization with chloroacetaldehyde followed by ultra-performance liquid chromatography with fluorescence detection. We used a 100 × 2.1 mm × 1.8 µm C18 column for the selective separation of analytes. Chromatographic separation was achieved with gradient elution of acetonitrile (flow rate 0.2 mL/min) from 2 to 20%. The eluent was initially composed of 10 mM KH2PO4 with 10 mM acetic acid and 25 µM heptafluorobutyric acid. The total analysis time was 11 min. Validation of the method included detection of the limit of detection (2 nM), limit of quantification (5 nM), accuracy (97.2-101%), and intra- and interday precision (2.2-9.0%). Analysis of plasma samples from healthy volunteers revealed that the average levels of S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine were 93.6, 20.9 and 14.8 nM, respectively.

High-performance liquid chromatography separation of the (S,S)- and (R,S)-forms of S-adenosyl-L-methionine.

S-adenosyl-l-methionine (AdoMet), an important biological cofactor, exists in two chiral forms, (S,S)- and (R,S)-, only the former of which is biologically active. Here, we have developed a chromatographic method to obtain pure (S,S)-AdoMet using a single C18 column.

Refolding of a fully functional flavivirus methyltransferase revealed that S-adenosyl methionine but not S-adenosyl homocysteine is copurified with flavivirus methyltransferase.

Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2'-O positions of the viral RNA cap by using S-adenosyl-l-methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by-product S-adenosyl-l-homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM-analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.

Fast quantification of S-adenosyl-L-methionine in dietary health products utilizing reversed-phase high-performance liquid chromatography: teaching an old method new tricks.

S-adenosyl-L-methionine is a ubiquitous methyl donor in living bodies. It is known to participate in several physiological processes including homocysteine metabolism and glutathione synthesis regulation, and cellular antioxidant mechanism. S-adenosyl-L-methionine containing dietary supplements has been prescribed recently for the treatment of depression, arthritis, and liver diseases with encouraging results. The development of an efficient analytical protocol for S-adenosyl-L-methionine containing dietary supplements is crucial for maintaining product quality and consumer health. In this study, the S-adenosyl-L-methionine content of several yeast products and commercial healthy food product samples was quantitatively analyzed utilizing HPLC. The chromatographic separation was achieved on a reversed-phase column and 2 % acetonitrile with a 98 % ammonium-acetate mobile phase under pH 4.5, with a flow rate of 1.0 mL/min. The wavelength used for detection with the UV detector was 254 nm. The total analysis time was short and the target compound showed a well-defined peak. The correlation coefficient of the regression curve showed good linearity and sensitivity with r = 0.999. All experiments were replicated five times and the relative standard deviations as well as the relative error values were all less than 3 %. Moreover, the achieved precision and accuracy values were high with 97.4-100.9 % recovery. Qualitative determination of S-adenosyl-L-methionine in the tested products was achieved using NMR and LC-MS techniques. The developed protocol is robust, fast, and suitable for the quality control analysis of yeast and commercial S-adenosyl-L-methionine products.

GenK-catalyzed C-6' methylation in the biosynthesis of gentamicin: isolation and characterization of a cobalamin-dependent radical SAM enzyme.

The existence of cobalamin (Cbl)-dependent enzymes that are members of the radical S-adenosyl-l-methionine (SAM) superfamily was previously predicted on the basis of bioinformatic analysis. A number of these are Cbl-dependent methyltransferases, but the details surrounding their reaction mechanisms have remained unclear. In this report we demonstrate the in vitro activity of GenK, a Cbl-dependent radical SAM enzyme that methylates an unactivated sp(3) carbon during the biosynthesis of gentamicin, an aminoglycoside antibiotic. Experiments to investigate the stoichiometry of the GenK reaction revealed that 1 equiv each of 5'-deoxyadenosine and S-adenosyl-homocysteine are produced for each methylation reaction catalyzed by GenK. Furthermore, isotope-labeling experiments demonstrate that the S-methyl group from SAM is transferred to Cbl and the aminoglycoside product during the course of the reaction. On the basis of these results, one mechanistic possibility for the GenK reaction can be ruled out, and further questions regarding the mechanisms of Cbl-dependent radical SAM methyltransferases, in general, are discussed.

Improved co-production of S-adenosylmethionine and glutathione using citrate as an auxiliary energy substrate.

The effects of sodium citrate on the fermentative co-production of S-adenosylmethionine (SAM) and glutathione (GSH) using Candida utilis CCTCC M 209298 were investigated. Sodium citrate was beneficial for the biosynthesis of SAM and GSH and in turn improved intracellular SAM and GSH contents. Adding 2 g/L of sodium citrate at 15 h was the most efficient approach for achieving elevated co-production of SAM and GSH. Using this sodium citrate addition mode, co-production of SAM and GSH reached 663.9 mg/L, which was increased by 27.5% compared to the control. Based on analysis of the kinetic parameters, evaluation of the energy metabolism and assay of key enzymes, sodium citrate was verified to act as an auxiliary energy substrate for the overproduction of SAM and GSH.

Determination of S-adenosyl-l-methionine in fruits by capillary electrophoresis.

INTRODUCTION: S-adenosyl-l-methionine (SAM) plays an important role in many biochemical reactions in plants. It is mainly used as a methyl donor for methylation reactions, but it also participates in, for example, the biosynthesis of polyamines and the plant hormone ethylene. OBJECTIVE: To develop a fast capillary electrophoresis technique to separate SAM in fruits and fruit juices without any pre-purification steps. METHODOLOGY: Four different extraction solutions and two extraction times were tested, of which 5% trichloroacetic acid (TCA) for 10 min was found most suited. A glycine : phosphate buffer (200 : 50 mm, pH 2.5) was found optimal to analyse SAM in TCA extracts. Analyses were preformed on different climacteric and non-climacteric fruits and fruit juices. The calibration curve was created in degraded tomato extract. The CE-method was compared with a more conventional HPLC method described in literature. RESULTS: The CE technique made it possible to completely separate the S,S- and R,S-diastereoisomeric forms of SAM. The CE method proved to be very fast (20 min total running time instead of 42 min) and more sensitive (limit of detection of 0.5 µm instead of 1 µm) compared with the conventional HPLC method. CONCLUSION: Fast measurements of SAM in fruits and juices are favoured by capillary electrophoresis in a 200 : 50 mm glycine : phosphate (pH 2.5) buffer system.

A fast and efficient method for quantitative measurement of S-adenosyl-L-methionine-dependent methyltransferase activity with protein substrates.

Modification of protein residues by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis-Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.

Oxygen vectors used for S-adenosylmethionine production in recombinant Pichia pastoris with sorbitol as supplemental carbon source.

In order to increase the yield of S-adenosylmethionine (SAM) in recombinant Pichia pastoris, a strategy of adding oxygen vectors and supplemental carbon sources was described. Three organic solutions were used as oxygen vectors for SAM accumulation at different concentrations and addition times. Firstly, n-hexane (0.5%) or n-heptane (1.0%) was added after 72 h of cultivation to improve SAM production. Carbon metabolism was scarce during the induction phase because of low methanol concentration. Secondly, sorbitol (1.2%), selected from three candidates (glycerol, lactic acid, and sorbitol), was used as the supplemental carbon source. The yield of SAM was improved significantly (53.26%) at 1.0%n-heptane added at 72 h (48 h induction), 1.2% sorbitol added at 72, 96, and 120 h of cultivation and 1.0% methanol added every 24 h during cultivation.

Enhancing yield of S-adenosylmethionine in Pichia pastoris by controlling NH4+ concentration.

Yield of S-adenosylmethionine was improved significantly in recombinant Pichia pastoris by controlling NH(4)(+) concentration. The highest production rate was 0.248 g/L h when NH(4)(+) concentration was 450 mmol/L and no repression of cell growth was observed. Within very short induction time (47 h), 11.63 g/L SAM was obtained in a 3.7 L bioreactor.

Simultaneous detection of S-adenosylmethionine and S-adenosylhomocysteine in mouse and rat tissues by capillary electrophoresis.

A capillary electrophoresis method for the determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in rat liver and kidney and mouse liver is described. The method can also be used to determine SAM in whole blood. The method provides rapid (approximately 16 min sample to sample) resolution of both compounds in perchloric extracts of tissues. Separation was performed by using an uncoated 50 microm ID capillary with 60 cm total length (50 cm to the detector window). Samples were separated at 22.5 kV and the separation running buffer was 200 mM glycine pH 1.8 (with HCl). The method compares favorably to HPLC methods (r (2) = 0.994 for SAM, r (2) = 0.998 for SAH) and has a mass detection limit of about 10 fmol for both SAM and SAH at a signal-to-noise ratio of 3. The method is linear over ranges of 1-100 microM SAM and 1-250 microM SAH. This method can be used to determine tissue concentrations of SAM and SAH, two metabolites that can provide insight into many biological processes.

[Isolation of S-adenosyl-L-methionine from an enriched Kluyveromyces lactis mutant, grown in whey media]. / Vydelenie S-adenozyl-L-metionin--obogashchennogo mutanta Kluyveromyces lactis, vyrashchivaemogo na srede s syvrotkoi.

The ability of six lactose-digesting yeast strains to accumulate S-adenosyl-L-methionine (AdoMet) when grown on whey medium was studied. Kluyveromyces lactis ATCC 8585 accumulated the greatest amount of AdoMet (15.2 mg per gram biomass) and was chosen for the development of an Ado-Met-enriched mutant. The Ado-Met-enriched mutant AM-65 was selected from mutants induced with N-methyl-N-nitro-N'-nitrosoguanidine. The strain accumulated 61.6 mg AdoMet per gram biomass. The effect of concentrations of medium components on AdoMet biosynthesis was studied. The ability of the mutant to accumulate AdoMet remained stable after multiple reinoculations.

HPLC analysis of S-adenosyl-L-methionine in pharmaceutical formulations.

A validated analytical method of S-adenosyl-L-methionine (SAM) in pharmaceutical preparations by reversed phase high performance liquid chromatography (RP-HPLC) is described. The compound is separated by a 2.1 mm x 15 cm, 5 microns--Discovery C18 column with isocratic elution. The effect of different anionic surface active agents with different molarity on the separation was studied. A direct relationship between the molarity of the surface active agents and the capacity factor (k') was found. The limit of detection was 0.49 mmol/ml and the linearity was r = 0.999 in the concentration range 20-100 micrograms/ml. Inter- and intra-assay variation was determined for three selected concentrations (20, 60, 100 micrograms/ml) by calculating the analytical recoveries with a range of 97.0-99.9%. The procedure was also suitable to check the stability of S-adenosyl-L-methionine in solution at room temperature.

Expression of the mRNA (N6-adenosine)-methyltransferase S-adenosyl-L-methionine binding subunit mRNA in cultured cells.

Ribonuclease protection assays (RPA) were used to detect and quantitate the amount of messenger RNA (mRNA) coding for the S-adenosyl-L-methionine binding subunit (MT-A70) of the mRNA (N6-adenosine)-methyltransferase from different types of cultured cells. HeLa cells cultured in suspension were analyzed at regular intervals along a normal growth curve. It was discovered that MT-A70 mRNA was transcribed constitutively across the time-course, irrespective of the rate of cellular proliferation. Further, 11 different cell lines representing non-tumorigenic, tumorigenic, and virally-transformed tumorigenic types from Homo sapiens, Mus musculus, and Rattus norvegicus were examined for MT-A70 mRNA expression. It was found that all the cell lines expressed a long and short splice-variant form of the gene. In general, the cell lines expressed a similar total amount of the MT-A70 mRNA while statistically significant differences existed between the quantity of the long and short forms among cell types. Tumorigenic cell lines synthesized as much as a 9-fold greater amount of long form versus short form MT-A70 mRNA. Comparatively, non-tumorigenic cell lines generally expressed only a 1.5-fold greater amount of long form versus short form MT-A70 mRNA.

A sensitive radioenzymatic assay for epinephrine forming enzymes.

Epinephrine (E) is formed in the adrenal medulla by phenylethanolamine-N-methyltransferase (PNMT), and in other tissues. Enzymes other than PNMT may be able to synthesize E, but this has been difficult to investigate because most assays do not have E as their final product. This assay produces 3H-E from norepinephrine (NE) and 3H-S-adenosylmethionine. The 3H-E is isolated on alumina, 3H-S-adenosylmethionine is precipitated and the 3H-E is suspended in diethylhexyl phosphoric acid in toluene for scintillation counting. The assay is sensitive and linear over a wide range. E was formed by most tissues tested. Brain and adrenal contained an enzyme specific for NE, but cardiac ventricle contained an enzyme that methylated both NE and dopamine. Denervated tissues in adrenal medullectomized rats contained very little NE, but still had E and E forming enzyme present. This assay detects a non-neuronal E forming enzyme with activity in vitro and in vivo.

Purification and characterization of some metabolic effects of S-neplanocylmethionine.

Our laboratory has previously demonstrated that treatment of mouse L929 cells with 1 microM neplanocin A results in the metabolic formation of S-neplanocylmethionine (Keller, B.T., and R.T. Borchardt, Biochem. Biophys. Res. Commun. 120:131-137 (1984]. The present study describes an efficient procedure for the purification of this analog from L cells based on its inherent chemical stability in alkaline conditions. Several metabolic effects of S-neplanocylmethionine are also reported. In L cells, S-neplanocylmethionine was determined to have an apparent half-life of 13 hr compared to 1 hr for S-adenosylmethionine during the initial 2 hr of a cycloleucine block. Analysis of polyamine levels in neplanocin A-treated cells showed a 3.8-fold decrease in putrescine and a 1.7-fold decrease in spermidine by 24 hr, reflecting a decrease in the cell growth rate in response to neplanocin A rather than a direct effect of S-neplanocylmethionine on the cellular S-adenosylmethionine decarboxylase. Consistent with these results are our findings that S-neplanocylmethionine does not significantly inhibit purified rat prostate or Escherichia coli S-adenosylmethionine decarboxylase and that [carboxy-14C]S-neplanocylmethionine exhibits no substrate activity with either enzyme. Purified S-neplanocylmethionine was observed to be a weak inhibitor of both S-adenosylmethionine-dependent protein carboxymethyltransferase and lipid methyltransferase in L cell extracts, having an IC50 value of 205 microM (S-adenosylmethionine = 10 microM). Similar studies with [methyl-3H]S-neplanocylmethionine indicate that the analog has little substrate activity in these two L cell methylation reactions and thus appears to act as a poor competitive inhibitor.

Enzymatic protein carboxyl methylation at physiological pH: cyclic imide formation explains rapid methyl turnover.

At pH 7.4, 37 degrees C, bovine brain protein carboxyl methyltransferase transiently methylates deamidated adrenocorticotropin. The methylation occurs at the alpha-carboxyl group of an atypical beta-carboxyl-linked isoaspartyl residue (position 25). Several lines of evidence indicate that the immediate product of demethylation is an aspartyl cyclic imide involving positions 25 and 26. The evidence includes (1) the rapid rate of methyl ester hydrolysis, which is consistent with intramolecular catalysis, (2) the inability of the demethylated product to be remethylated, (3) the charge of this product, and (4) its rate of breakdown. The eventual hydrolysis of the cyclic imide produces a 30/70 mixture of peptides containing either alpha- or beta-carboxyl-linked aspartyl residues, respectively. Cyclic imide formation is nonenzymatic and can explain the unusual lability of mammalian protein methyl esters in general. These findings suggest that protein carboxyl methylation in mammalian tissues is not a simple on/off reversible modification as it apparently is in chemotactic bacteria. Carboxyl methylation may serve to activate selected protein carboxyl groups for subsequent longer lasting modifications, possibly subserving a role in protein repair, degradation, cross-linking, or some other as yet undiscovered alteration of protein structure.

Purification and properties of S-adenosylmethionine: aldoxime O-methyltransferase from Pseudomonas sp. N.C.I.B. 11652.

An enzyme catalysing the O-methylation of isobutyraldoxime by S-adenosyl-L-methionine was isolated from Pseudomonas sp. N.C.I.B. 11652. The enzyme was purified 220-fold by DEAE-cellulose chromatography, (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and chromatography on calcium phosphate gel. Homogeneity of the enzyme preparation was confirmed by isoelectric focusing on polyacrylamide gel and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme showed a narrow pH optimum at 10.25, required thiol-protecting agents for activity and was rapidly denatured at temperatures above 35 degrees C. The Km values for isobutyraldoxime and S-adenosyl-L-methionine were respectively 0.24 mM and 0.15 mM. Studies on substrate specificity indicated that attack was mainly restricted to oximes of C4-C6 aldehydes, with preference being shown for those with branching in the 2- or 3-position. Ketoximes were not substrates for the enzyme. Gel filtration on Sephadex G-100 gave an Mr of 84 000 for the intact enzyme, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated an Mr of 37 500, suggesting the presence of two subunits in the intact enzyme. S-Adenosylhomocysteine was a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 0.027 mM. The enzyme was also susceptible to inhibition by thiol-blocking reagents and heavy-metal ions. Mg2+ was not required for maximum activity.

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

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