A deuterium surface coil NMR study of the metabolism of D-methionine in the liver of the anesthetized rat

The hepatic metabolism of deuteriated D-methionine has been studied in the intact, anesthetized rat using 2H NMR spectroscopy. The rate of formation of the principal labeled metabolite, [methyl-2H3]sarcosine, from the D-[methyl-2H3]methionine precursor was found to be as rapid as the rate observed previously in NMR studies of the hepatic metabolism of L-methionine. Similarly, rates of clearance of labeled methionine from the liver, formation of N-trimethyl-labeled metabolites, and labeling of the HDO pool were all found to be similar to the rates observed in the L-methionine studies. In contrast, all of these metabolic transformations are strongly inhibited by pretreatment of the rats with sodium benzoate, an inhibitor of D-amino acid oxidase. In vivo 2H NMR studies of sodium benzoate treated rats given L-[methyl-2H3]-methionine exhibit a much more rapid formation of [methyl-2H3]sarcosine than rats given the D enantiomer, consistent with the expectation that the sodium benzoate does not interfere with either the formation of S-adenosylmethionine or the subsequent transmethylation of glycine. However, the rates of methionine clearance and formation of deuteriated water are markedly reduced in this study relative to rats receiving the labeled D- or L-methionine without sodium benzoate pretreatment. These results indicate that subsequent to the initial oxidative deamination of the labeled D-methionine, the reamination to give L-methionine is rapid compared with the further degradation of the alpha-keto acid. Thus, the results are consistent with a dominant contribution of the glycine/sarcosine shuttle to the metabolism of excess D- or L-methionine.     

Quantitative evaluation and regulation of S-adenosylmethionine-dependent transmethylation in sheep tissues

The overall rates of S-adenosylmethionine (AdoMet)-dependent transmethylation were estimated in various tissues from the initial rate of S-adenosylhomocysteine (AdoHcy) plus AdoMet accumulation after blocking hydrolysis of AdoHcy. The rates were found to differ widely among the tissues of sheep and the highest rate was in the pancreas, being 600 times higher than that in the muscle. Sheep liver possessed approximately 75% of total-body capacity for transmethylation although the transmethylation rate was approximately half that in rat liver. The minimum estimate of daily requirement of AdoMet for transmethylation for adult sheep was approximately 18 mmol, far in excess of methionine intake. Methionine loading elevated AdoMet levels only in the tissues with a high or moderate rate of transmethylation. The kinetic properties of major methyltransferases in sheep liver along with tissue distribution of AdoMet and AdoHcy suggest that transmethylation rate is subject to physiological regulation by tissue levels of AdoMet and AdoHcy.     

Selective reversible deuteriation of oligodeoxynucleotides: simplification of two-dimensional nuclear Overhauser effect NMR spectral assignment of a non-self-complementary dodecamer duplex

Oligodeoxynucleotides are reversibly deuteriated at the purine C8 and cytosine C5 positions with deuterioammonium bisulfite at pD 7.8. The exchange reaction is complete after 48 h at 65 degrees C. When an oligomer deuteriated under these conditions is analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy, the purine H8 and cytosine H5 proton signals are selectively removed from the spectrum. A non-self-complementary oligodeoxynucleotide that has been deuteriated in this manner may be annealed with its complement and the resulting heteroduplex analyzed by two-dimensional nuclear Overhauser enhancement (NOESY) spectroscopy. NOE cross-peaks arising from pyrimidine H6-deoxyribose H1' dipolar interactions in both strands are observed, but purine H8-deoxyribose H1' and purine H8-deoxyribose H2',H2" dipolar interactions are only observed for the nondeuteriated strand. The intense cytosine H5-H6 cross-peaks are also removed from the spectrum of the deuteriated strand, which further simplifies interpretation since these strong cross-peaks often interfere with less intense NOE cross-peaks arising from dipolar coupling between purine H8 or pyrimidine H6 and deoxyribose anomeric protons. The resulting spectral simplification allows unambiguous assignments to be made on NOEs that otherwise may be difficult to distinguish. The deuteration procedure is demonstrated with the sequence d(CGTTATAATGCG).d(CGCATTATAACG), which has previously been assigned by traditional NOESY methods [Wemmer, D. E., Chou, S.-H., Hare, D. R., & Reid, B. R. (1984) Biochemistry 23, 2262-2268]. Although the assignment of this dodecadeoxynucleotide may be completed without deuteriation, several NOEs must be assigned indirectly because of degeneracies in the chemical shift of the purine H8 protons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevated overall rates of transmethylation in cell lines from diverse human tumors

Summary In a study of a diverse set of human tumor cell lines previously shown to all have a defect in methionine metabolism (Stern, P. H., Wallace, C. D. and Hoffman, R. M. J. Cellular Physiology119, 29–34, 1984), we demonstrate in this report that all have enhanced overall rates of transmethylation compared to normal human fibroblasts. Transmethylation rates were measured by blocking S-adenosylhomocysteine hydrolase and measuring the AdoHcy which accumulates as a result of transmethylation. The enhanced transmethylation rates may be the basis of the above-mentioned defects in methionine metabolism previously reported in human tumor cells, including the basis of the inability of the majority of the tumor cells to grow when methionine is replaced by homocysteine. The excess and unbalanced tRNA methylation observed for the last 25 years in many types of cancer may be at least in part explained by our results of elevated rates of overall transmethylation in cancer cells. The alteration of such a fundamental process as transmethylation in cancer may be indicative of its importance in the oncogenic process. This study was supported by grants 1348A and 1496R1 from the Council for Tobacco Research-USA, Inc., grant CA27564 from the National Cancer Institute, and Research Career Development Award CA00804 from the National Cancer Institute, all to Robert M. Hoffman, and by the George A. Jacobs Memorial Fund for Cancer Research. Editor's Statement This report describes increased rates of transmethylation in a large number of human tumor cell lines in culture, compared to transmethylation rates of several strains of untransformed human fibroblasts. All studies of this kind, using tumor cell lines of epithelial origin and employing as controls “normal” (untransformed) cell strains that are solely of fibroblastic origin, are difficult to interpret and remain open to question. However, the authors' observations that cell lines derived from both sarcomas and carcinomas exhibit enhanced transmethylation rates may strengthen, the case somewhat. More importantly, the potential relationship discussed by the authors of enhanced transmethylation rates to the phenomena of methionine dependence and unbalanced tRNA methylation make the data presented worthy of note. Gordon H. Sato     

Measurement of intracellular sulfur amino acid metabolism in humans

Methionine metabolism forms homocysteine via transmethylation. Homocysteine is either 1) condensed to form cystathionine, which is cleaved to form cysteine, or 2) remethylated back to methionine. Measuring this cycle with the use of isotopically labeled methionine tracers is problematic, because the tracer is infused into and measured from blood, whereas methionine metabolism occurs inside cells. Because plasma homocysteine and cystathionine arise from intracellular metabolism of methionine, plasma homocysteine and cystathionine enrichments can be used to define intracellular methionine enrichment during an infusion of labeled methionine. Eight healthy, postabsorptive volunteers were given a primed continuous infusion of [1-13C]methionine and [methyl-2H(3)]methionine for 8 h. Enrichments in plasma methionine, [13C]homocysteine and [13C]cystathionine were measured. In contrast to plasma methionine enrichments, the plasma [13C]homocysteine and [13C]cystathionine enrichments rose to plateau slowly (rate constant: 0.40 +/- 0.03 and 0.49 +/- 0.09 h(-1), respectively). The enrichment ratios of plasma [13C]homocysteine to [13C]methionine and [13C]cystathionine to [13C]methionine were 58 +/- 3 and 54 +/- 3%, respectively, demonstrating a large intracellular/extracellular partitioning of methionine. These values were used to correct methionine kinetics. The corrections increase previously reported rates of methionine kinetics by approximately 40%.     

Incorporation of labeled formate in rat liver mitochondrial proteins and initiation of protein synthesis

During incubation of rat liver mitochondria in vitro labeled formate is incorporated into TCA-insoluble substance during mitochondrial translation. Data from hydrolysis with CNBr (after methionine residues) or with 0.5 N HCl (deformylation of amino acid N-formyl derivatives) suggest that about half of the total protein radioactivity is incorporated in formate groups of N-terminal methionine. Labeling of growing polypeptides with formate (but not with phenylalanine or methionine) oscillates with a period of about 13 min. The potential initiation capacity is unchangeable and exceeds that observed experimentally by one order of magnitude. The data obtained are consistent with the previously proposed hypothesis no synchronization of mitochondrial protein synthesis which cannot be induced by the steps preceding the formation of the first peptide bound.     

Assay of the concentration and (13)C isotopic enrichment of propionyl-CoA,methylmalonyl-CoA,and succinyl-CoA by gas chromatography-mass spectrometry

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [(2)H(5)]propionyl-CoA and [(2)H(4)]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-(13)C(3)]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine.     

Localization and identification of covalently bound flavoproteins in rat liver mitochondria by prelabeling of their flavin moiety

The covalently bound flavoproteins in rat liver mitochondria were prelabeled by injecting [14C]riboflavin into a rat, then liver mitochondria were obtained and further labeled with [3H]pargyline, a suicide inhibitor of monoamine oxidase. When the mitochondria were subjected to osmotic lysis, two covalently bound flavoproteins having molecular weights of 110,000 and 94,000 were found in the supernatant. These proteins were identified as sarcosine dehydrogenases. Upon treatment of the membranous fraction with 1% Triton X-100, succinate dehydrogenase with a molecular weight of 70,000 was found in the soluble fraction, while two well-separated proteins doubly-labeled with 14C and 3H were found in the insoluble fraction. Their molecular weights were 61,000 and 57,000. By isoelectric focusing, two 3H peaks were observed with pI values of 8.3 and 8.4. The former corresponded to the 61,000-dalton protein, and the latter, to the 57,000 one. From the data obtained by using selective inhibitors, deprenyl and clorgyline, the [3H]pargyline-binding proteins with molecular weights of 61,000 and 57,000 were assigned to proteins of monoamine oxidases of type A and type B, respectively.     

Insulin in methionine and homocysteine kinetics in healthy humans: plasma vs. intracellular models

Methionine is a sulfur-containing amino acid that is reversibly converted into homocysteine. Homocysteine is an independent cardiovascular risk factor frequently associated with the insulin resistance syndrome. The effects of insulin on methionine and homocysteine kinetics in vivo are not known. Six middle-aged male volunteers were infused with L-[methyl-2H3,1-13C]methionine before (for 3 h) and after (for 3 additional hours) an euglycemic hyperinsulinemic (150 mU/l) clamp. Steady-state methionine and homocysteine kinetics were determined using either plasma (i.e., those of methionine) or intracellular (i.e., those of plasma homocysteine) enrichments. By use of plasma enrichments, insulin decreased methionine rate of appearance (Ra; both methyl- and carbon Ra) by 25% (P < 0.003 vs. basal) and methionine disposal into proteins by 50% (P < 0.0005), whereas it increased homocysteine clearance by approximately 70% (P < 0.025). With intracellular enrichments, insulin increased all kinetic rates, mainly because homocysteine enrichment decreased by approximately 40% (P < 0.001). In particular, transmethylation increased sixfold (P < 0.02), transsulfuration fourfold (P = 0.01), remethylation eightfold (P < 0.025), and clearance eightfold (P < 0.004). In summary, 1) physiological hyperinsulinemia stimulated homocysteine metabolic clearance irrespective of the model used; and 2) divergent changes in plasma methionine and homocysteine enrichments were observed after hyperinsulinemia, resulting in different changes in methionine and homocysteine kinetics. In conclusion, insulin increases homocysteine clearance in vivo and may thus prevent homocysteine accumulation in body fluids. Use of plasma homocysteine as a surrogate of intracellular methionine enrichment, after acute perturbations such as insulin infusion, needs to be critically reassessed.     

Kinetics of labeling of the S-adenosylmethionine pool of Saccharomyces cerevisiae.

A method has been developed for studying the specific activity of the pool of S-adenosylmethionine in yeast. The pool reaches half-maximal specific activity within 30 s after the addition of [methyl-3H]methionine. After addition of an excess of nonradioactive methionine, the specific activity of S-adenosylmethionine is reduced by half within 20 s. During that period there is a substantial expansion of the pool. A logarithmically growing cell in synthetic medium contains about 2 X 10(6) molecules of S-adenosylmethionine, of which only 10% is used for the methylation of ribonucleic acid molecules.     

Synthesis and targeting of hexokinase to mitochondria in hepatoma cells

The synthesis and turnover of hexokinase has been measured in Zajdela hepatoma ascites cells labeled for short periods with [35S]methionine. Digitonin fractionation of the labeled cells into a soluble and a membrane fraction showed that only a small part of the newly labeled hexokinase is transferred to mitochondrial binding sites. The soluble enzyme disappears, however, with a half-life of less than 2 h. Glucose had no effect on the stability of the soluble enzyme in intact cells. Our experiments suggest that Zajdela cell hexokinase is synthesized in excess of binding sites and that the excess enzyme is not stable.     

Histone H3 lysine-trimethylation markers are decreased by recombinant methioninase and increased by methotrexate at concentrations which inhibit methionine-addicted osteosarcoma cell proliferation

Methionine addiction is a fundamental and general hallmark of cancer cells, which require exogenous methionine, despite their ability to synthesize normal amounts of methionine from homocysteine. In contrast, methionine-independent normal cells do not require exogenous methionine in the presence of a methionine precursor. The methionine addiction of cancer cells is due to excess transmethylation reactions. We have previously shown that histone H3 lysine marks are over-methylated in cancer cells and the over-methylation is unstable when the cancer cells are restricted of methionine. In the present study, we show that methionine-addicted osteosarcoma cells are sensitive to both methotrexate (MTX) and recombinant methioninase (rMETase), but they affect histone H3 lysine-methylation in the opposite direction. Concentrations of MTX and rMETase, which inhibit osteosarcoma cells viability to 20%, had opposing effects on the status of histone methylation of H3K9me3 and H3K27me3. rMETase significantly decreased the amount of H3K9me3 and H3K27me3. In contrast, MTX significantly increased the amount of H3K9me and H3K27me3. The results suggest that increase or decrease in these methylated histone lysine marks is associated with proliferation arrest of methionine-addicted osteosarcoma.     

Regulation of species-specific ribosomal RNA in a somatic hybrid cell.

When 13B hamster-mouse hybrid cells are harvested either right after 4 h of incubation with [me-3H]methionine or following 26 h of "chase" with excess non-radioactive methionine, in both cases about half of the labeled cytoplasmic rRNA is of hamster type. It had been previously shown in this laboratory (Eliceiri, G.L. (1973) Biochim. Biophys. Acta 312, 737-741) that when [3H]uridine was the radioactive precursor about 80% of the labeled cytoplasmic rRNA was of hamster type after a short incubation, and about half after a long incubation. It is postulated that a temporary difference in the specific acitivity of [3H]UTP in possibly segregated mouse and hamster types of nucleoli might account for these results. The master/mouse ratio of cytoplasmic rRNA in hybrid 13B is similar in free and in membrane-bound ribosomes, and in ribosomes of sparse (rapidly growing) cell populations and of confluent (slowly growing) cells.     

Brain Protein Synthesis in the Conscious Rat Using L-[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways

The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady-state conditions, the specific activity of L-[35S] methionine in the tRNA-bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L-[35S]-Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.     

Cloning and Mapping of the cDNA for Human Sarcosine Dehydrogenase, a Flavoenzyme Defective in Patients with Sarcosinemia

Sarcosine dehydrogenase is a liver mitochondrial matrix flavoenzyme that is defective in patients with sarcosinemia, a rare autosomal metabolic defect characterized by elevated levels of sarcosine in blood and urine. Some patients also exhibit mental retardation and growth failure. A full-length cDNA for human sarcosine dehydrogenase was isolated from an adult liver cDNA library. The first 22 residues in the deduced amino acid sequence exhibit features expected for a mitochondrial targeting sequence. The predicted mass of the mature human liver sarcosine dehydrogenase (99,505 Da) is in good agreement with that observed for rat liver sarcosine dehydrogenase ( approximately 100,000 Da). Human sarcosine dehydrogenase exhibits 89% identity with rat liver sarcosine dehydrogenase and strong homology ( approximately 35% identity) with rat liver dimethylglycine dehydrogenase, a sarcosine dehydrogenase-related protein from Rhodobacter capsulatus, and the regulatory subunit from bovine pyruvate dehydrogenase phosphatase. The human sarcosine dehydrogenase gene is at least 75.3 kb long and located on chromosome 9q34. The adult human liver clone is assembled from 21 exons (1-6, 7a, 8a, 9-21). Two smaller cDNA clones, isolated from adult liver and infant brain libraries, were assembled from the same sarcosine dehydrogenase gene by the use of alternate polyadenylation and splice sites. This is the first report of the genomic structure of the sarcosine dehydrogenase gene in any species. The observed chromosomal location is consistent with genetic studies with a mouse model for sarcosinemia that map the mouse gene to a region of mouse chromosome 2 syntenic with human 9q33-q34. The availability of the SDH gene sequence will enable characterization of the genotypes of sarcosinemia patients with different phenotypes.     

Biosynthesis of tritium-labeled S-adenosyl-L-methionine in yeast cells

Biosynthetic preparation of S-adenosyl-L-[methyl-3H]methionine from L-[methyl-3H]methionine by cultivation of diploid yeast Saccharomyces cerevisiae (methionine-auxotrophic) in a cultural medium with the high concentration of L-methionine is described. The radiochemical purity was over 95%. Biological activity of the preparations has been shown in transmethylation reactions in the presence of the yeast homocysteine-methyltransferase.     

Glyphosate catabolism by Pseudomonas sp. strain PG2982.

The pathway for the degradation of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. PG2982 has been determined by using metabolic radiolabeling experiments. Radiorespirometry experiments utilizing [3-14C]glyphosate revealed that approximately 50 to 59% of the C-3 carbon was oxidized to CO2. Fractionation of stationary-phase cells labeled with [3-14C]glyphosate revealed that from 45 to 47% of the assimilated label is distributed to proteins and that the amino acids methionine and serine are highly labeled. Adenine and guanine received 90% of the C-3 label found in the nucleic acid fraction, and the only pyrimidine base labeled was thymine. These results indicated that C-3 of glyphosate was at some point metabolized to a C-1 compound whose ultimate fate could be both oxidation to CO2 and distribution to amino acids and nucleic acid bases that receive a C-1 group from the C-1-donating coenzyme tetrahydrofolate. Pulse-labeling of PG2982 cells with [3-14C]glyphosate resulted in the isolation of [3-14C]sarcosine as an intermediate in glyphosate degradation. Examination of crude extracts prepared from PG2982 cells revealed the presence of a sarcosine-oxidizing enzyme that oxidizes sarcosine to glycine and formaldehyde. These results indicate that the first step in glyphosate degradation by PG2982 is cleavage of the carbon-phosphorus bond, resulting in the release of sarcosine and a phosphate group. The phosphate group is utilized as a source of phosphorus, and the sarcosine is degraded to glycine and formaldehyde. This pathway is supported by the results of [1,2-14C]glyphosate metabolism studies, which show that radioactivity in the proteins of labeled cells is found only in the glycine and serine residues.     

Uptake and processing of liposomal phospholipids by Kupffer cells in vitro

We investigated the intracellular metabolic fate of [Me-14C]choline-labeled phosphatidylcholines and sphingomyelin taken up by rat Kupffer cells in maintenance culture during interaction with large unilamellar liposomes composed of cholesterol, labeled choline-phospholipid and phosphatidylserine (molar ration 5:4:1). With both labeled compounds only small proportions of water-soluble radioactivity were found to accumulate in the cells and in the culture medium, suggesting limited phospholipid degradation. However, after a lag period of 30 min progressively increasing proportions of cell-associated liposomal phospholipid were found to be converted to cellular phospholipid, nearly all of which was phosphatidylcholine. This conversion as well as the limited release of water-soluble label from the cells was inhibited by the lysosomotropic agents ammonium chloride and chloroquine. With [Me-14C]choline-labeled lysophosphatidylcholine, label was found to become cell-associated far in excess of an encapsulated liposomal label, [3H]inulin. Without a lag period virtually all of this was rapidly converted to phosphatidylcholine, a process which was not inhibited by the lysosomotropic agents. It is concluded that Kupffer cells, after endocytosis of liposomes, degrade the liposomal phospholipids effectively but reutilize the choline moiety for de novo synthesis of cellular phosphatidylcholine.     

Regioselective synthesis of 1I,1II,5I,5II,6I,6I,6II,6II-2H8-cellobiose

Partially deuteriated 1,5,6,6-(2)H(4)-d-glucose and 1(I),1(II),5(I),5(II),6(I),6(I),6(II),6(II)-(2)H(8)-d-cellobiose were synthesized in high yields and on a large scale from d-glucose. (2)H enrichment at C-5 and C-6 of each glucopyranosyl unit in excess of 85% and 90%, respectively, was realized by (1)H-(2)H exchange in (2)H(2)O containing deuteriated Raney Ni. Nucleophilic addition of LiAlD(4) to 5,6,6-(2)H(3)-2,3,4,6-tetra-O-benzyl-d-gluconolactone led to a 98% (2)H enrichment at C-1. Deuteriated cellobiose is of interest as building block for the synthesis of a model compound of cellulose I.     

Two distinct site-dependent biosynthetic pathways for the incorporation of sarcosine into actinomycins.

Actinomycins II and III, containing sarcosine residues in two adjacent sites of their peptide moieties were produced by Streptomyces antibioticus in the presence of exogenous sarcosine labeled with deuterium in the N-methyl group. Combined gas chromatography-mass spectrometry of the cyclodipeptides derived by thermal degradation of these actinomycins demonstrated specific incorporation of the labeled sarcosine into the 3-site, implicating some other biosynthetic precursor, presumably glycine, for the sarcosine in the 4-site. The same conclusion emerged from proton nuclear magnetic resonance spectroscopy of these deuterium-labeled actinomycins.