S-adenosylmethionine Biosynthesis Research Articles

Ethanol Mediated Loss Of MAT2A (Methionine Adenosylmethyltransferase) Expression And Same (Sadenosylmethionine) Biosynthesis Induce Fas-mediated Cd4+ T Lymphocyte Death: Potential Mechanism(s) For Alcohol Mediated Immunotoxicity

Ethanol Mediated Loss Of MAT2A (Methionine Adenosylmethyltransferase) Expression And Same (Sadenosylmethionine) Biosynthesis Induce Fas-mediated Cd4+ T Lymphocyte Death: Potential Mechanism(s) For Alcohol Mediated Immunotoxicity

Role of methionine adenosyltransferase and S-adenosylmethionine in alcohol-associated liver cancer

Two genes ( MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT), which catalyzes the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and, in the liver, a precursor of glutathione. MAT1A is expressed mostly in the liver, whereas MAT2A is widely distributed. MAT2A is induced in the liver during periods of rapid growth and dedifferentiation. In human hepatocellular carcinoma (HCC) MAT1A is replaced by MAT2A. This is important pathogenetically because MAT2A expression is associated with lower SAMe levels and faster growth, whereas exogenous SAMe treatment inhibits growth. Rats fed ethanol intragastrically for 9 weeks also exhibit a relative switch in hepatic MAT expression, decreased SAMe levels, hypomethylation of c- myc, increased c- myc expression, and increased DNA strand break accumulation. Patients with alcoholic liver disease have decreased hepatic MAT activity owing to both decreased MAT1A expression and inactivation of the MAT1A-encoded isoenzymes, culminating in decreased SAMe biosynthesis. Consequences of chronic hepatic SAMe depletion have been examined in the MAT1A knockout mouse model. In this model, the liver is more susceptible to injury. In addition, spontaneous steatohepatitis develops by 8 months, and HCC develops by 18 months. Accumulating evidence shows that, in addition to being a methyl donor, SAMe controls hepatocyte growth response and death response. Whereas transient SAMe depletion is necessary for the liver to regenerate, chronic hepatic SAMe depletion may lead to malignant transformation. It is interesting that SAMe is antiapoptotic in normal hepatocytes, but proapoptotic in liver cancer cells. This should make SAMe an attractive agent for both chemoprevention and treatment of HCC.

Induction of Human Methionine Adenosyltransferase 2A Expression by Tumor Necrosis Factor α: ROLE OF NF-κB AND AP-1

Two genes (MAT1A and MAT2A) encode for methionine adenosyltransferase (MAT), an essential cellular enzyme responsible for S-adenosylmethionine biosynthesis. MAT1A is expressed mostly in the liver, whereas MAT2A is widely distributed. We showed a switch from MAT1A to MAT2A expression in human hepatocellular carcinoma (HCC), which facilitates cancer cell growth. Using DNase I footprinting analysis, we previously identified a region in the MAT2A promoter protected from DNase I digestion in HCC. This region contains NF-kappa B and AP-1 elements, and the present study examined whether they regulate MAT2A promoter activity. We found nuclear binding of NF-kappa B and AP-1 to the MAT2A promoter increased in HCC. Tumor necrosis factor alpha (TNFalpha), which activates both NF-kappa B and AP-1, increased MAT2A expression in a dose- and time-dependent manner, binding of both NF-kappa B and AP-1 to the MAT2A promoter and MAT2A promoter activity, with the latter effect blocked by site-directed mutagenesis of the NF-kappa B and AP-1 binding sites. Blocking NF-kappa B with I kappa B super-repressor or AP-1 with dominant-negative c-Jun led to decreased basal MAT2A expression and prevented the TNF alpha-induced increase in MAT2A expression. Although blocking NF-kappa B had no influence on the ability of TNF alpha to increase AP-1 nuclear binding, blocking AP-1 with dominant-negative c-Jun prevented the TNF alpha-mediated increase in NF-kappa B binding. In conclusion, both NF-kappa B and AP-1 are required for basal MAT2A expression in HepG2 cells and mediate the increase in MAT2A expression in response to TNF alpha treatment. Increased trans-activation of these two sites also contributes to MAT2A up-regulation in HCC.

Regulation of the Human MAT2B Gene Encoding the Regulatory β Subunit of Methionine Adenosyltransferase, MAT II

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet), a key molecule in transmethylation reactions and polyamine biosynthesis. The MAT II isozyme consists of a catalytic alpha2 and a regulatory beta subunit. Down-regulation of the MAT II beta subunit expression causes a 6-10-fold increase in intracellular AdoMet levels. To understand the mechanism by which the beta subunit expression is regulated, we cloned the MAT2B gene, determined its organization, characterized its 5'-flanking sequences, and elucidated the in vitro and in vivo regulation of its promoter. Transcription of the MAT2B gene initiates at position -203 relative to the translation start site. Promoter deletion analysis defined a minimal promoter between positions +52 and +93 base pairs, a GC-rich region. Inclusion of the sequences between -4 and +52 enhanced promoter activity; this was primarily because of an Sp1 recognition site at +9/+15. The inclusion of sequences up to position -115 provided full activity; this was attributed to a TATA at -32. The Sp1 site at position +9 was key for the formation of protein.DNA complexes. Mutation of both the Sp1 site at +9 and the TATA at -32 reduced promoter activity to its minimal level. Supershift assays showed no effect of the anti-Sp1 antibody on complex formation, whereas the anti-Sp3 antibody had a strong effect on protein.DNA complex formation, suggesting that Sp3 is one of the main factors binding to this Sp1 site. Chromatin immunoprecipitation assays supported the involvement of both Sp1 and Sp3 in complexes formed on the MAT2B promoter. The data show that the 5'-untranslated sequences play an important role in regulating the MAT2B gene and identifies the Sp1 site at +9 as a potential target for modulating MAT2B expression, a process that can have a major effect on intracellular AdoMet levels.

DNA methylation and histone acetylation of rat methionine adenosyltransferase 1A and 2A genes is tissue-specific

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine (AdoMet). In mammals MAT activity derives from two separate genes which display a tissue-specific pattern of expression. While MAT1A is expressed only in the adult liver, MAT2A is expressed in non-hepatic tissues. The mechanisms behind the selective expression of these two genes are not fully understood. In the present report we have evaluated MAT1A and MAT2A methylation in liver and in other tissues, such as kidney, by methylation-sensitive restriction enzyme digestion of genomic DNA. Our data indicate that MAT1A is hypomethylated in liver and hypermethylated in non-expressing tissues. The opposite situation is found for MAT2A. Additionally, histones associated to MAT1A and MAT2A genes showed enhanced levels of acetylation in expressing tissues (two-fold for MAT1A and 3.5-fold for MAT2A liver and kidney respectively). These observations support a role for chromatin structure and its modification in the tissue-specific expression of both MAT genes.

Methionine Adenosyltransferase Activity in Erythrocytes and Spinal Cord of Patients with Sporadic Amyotrophic Lateral Sclerosis

The role of transmethylation mechanisms in the etiology of amyotrophic lateral sclerosis (ALS) is hitherto unexplored. The activity of l-methionine S-adenosyltransferase (MAT), a regulatory enzyme of S-adenosylmethionine biosynthesis, was investigated in erythrocytes of 21 patients with ALS, spinal cord specimens of 7 ALS patients, and matched controls. In ALS patients the activity of MAT in erythrocytes was sex-dependent. In comparison with controls, the male group presented a 33% higher Vmax (P < 0.05) and a 41% decrease in the affinity of MAT for methionine (Km, P < 0.05). The type of ALS onset (limb or bulbar), age, or duration of the disease did not influence erythrocyte MAT activity. In the spinal cord, the activity of MAT was homogeneously distributed through dorsal horn, ventral horn, and white matter. Comparisons between data from controls and ALS patients and analysis of sex effect showed no significant differences. The kinetic difference of erythrocyte MAT in the male group of ALS patients might be interesting to explore since it is well known that there is a male predominance of 1.5 to 2.5:1 in ALS.

Characterization of Rat Liver-specific Methionine Adenosyltransferase Gene Promoter: ROLE OF DISTAL UPSTREAM cis-ACTING ELEMENTS IN THE REGULATION OF THE TRANSCRIPTIONAL ACTIVITY

Methionine adenosyltransferase is a ubiquitous enzyme that catalyzes the only known route of biosynthesis of S-adenosylmethionine, the major methyl group donor in cell metabolism. In mammals, two different methionine adenosyltransferases exist: one is confined to the liver, and the other one is distributed in extrahepatic tissues. In the present study, we report the cloning of the 5'-flanking region of liver-specific methionine adenosyltransferase gene from rat. Two closely spaced sites for transcriptional initiation were identified by primer extension analysis. The major transcription start site was determined to be 29 nucleotides downstream from the putative TATA box. Transient transfection assays of constructs containing sequentially deleted 5'-flanking sequences fused to the luciferase gene showed that rat hepatic methionine adenosyltransferase promoter was able to efficiently drive reporter expression not only in liver-type cells (rat hepatoma H35 cells and human hepatoblastoma HepG2 cells) but also in Chinese hamster ovary cells. Two regions spanning nucleotides -1251 to -958 and -197 to +65 were found to be crucial for the promoter efficiency. The distal upstream region contains elements that positively regulate promoter activity in H35 and HepG2 cells but are ineffective in Chinese hamster ovary cells. Eight protein binding sites were characterized in both regions by DNase I footprinting analysis. Two of these elements, sites A and B, located in the distal region, were found to be essential for the regulation of promoter activity. Electrophoretic mobility shift assays and competition experiments showed that site A is recognized by an NF1 protein. Site B was able to interact with a member of HNF-3 family when nuclear extracts from rat liver and H35 cells were used in the in vitro assay, but an additional binding activity to an NHF1-like protein was obtained with the hepatoma cell extracts. It is suggested that this differential binding can contribute to the cell specificity of promoter function.

Transport ofS-Adenosylmethionine in Isolated Rat Liver Mitochondria

Mitochondria do not have the enzyme, methionine adenosyltransferase (ATP:l-methionineS-adenosyltransferase, EC 2.5.1.6), necessary for the biosynthesis ofS-adenosylmethionine. Nevertheless, about 30% of total hepaticS-adenosylmethionine resides in the mitochondria and radiolabeledS-adenosylmethionine may be isolated from the mitochondria after administration of radiolabeled methionine. This leads to the hypothesis that a carrier-mediated system is responsible forS-adenosylmethionine transport from the cytosol into the mitochondria. We have characterized such a system in isolated rat liver mitochondria. Uptake ofS-adenosylmethionine consisted of two components. One component was incorporation of the methyl group into phospholipids as shown by thin-layer chromatography. The second component represented uptake into the mitochondria since addition of excess unlabeledS-adenosylmethionine resulted in efflux of labeled substrate. This countertransport is characteristic of a carrier-mediated transport system. Uptake (corrected for incorporation into phospholipids) was saturable with an apparentKm= 8.9 μmandVmax= 54.3 pmol·mg protein−1·min−1. Uptake was not inhibited by methionine, adenosine, 5′-methylthioadenosine, carnitine, choline, betaine, quinine, or hemicholinium-3. Uptake was inhibited by sinefungin and byS-adenosylhomocysteine (Ki= 53.4 μm). Uptake ofS-adenosylmethionine was not dependent on the electrical potential across the mitochondrial membrane. These results indicate thatS-adenosylmethionine is taken up into mitochondria via a specific, carrier-mediated system.

Betaine, ethanol, and the liver: A review

Two of the most important biochemical hepatic pathways in the liver are those that synthesize methionine and S-adenosylmethionine (SAM) through the methylation of homocysteine. This article reviews some recent findings in this laboratory, which demonstrate that ethanol feeding to rats impairs one of these pathways involving the enzyme methionine synthetase (MS), but by way of compensation increases the activity of the enzyme betaine:homocysteine methyl transferase (BHMT), which catalyzes the second pathway in methionine and SAM biosynthesis. It has been shown that despite the inhibition of MS, the enhanced BHMT pathway utilizes hepatic betaine pools to maintain levels of SAM. Subsequent to the above findings, it has been shown that minimal supplemental dietary betaine at the 0.5% level generates SAM twofold in control animals and fivefold in ethanol-fed rats. Concomitant with the betaine-generated SAM, ethanol-induced hepatic fatty infiltration was ameliorated. In view of the fact that SAM has already been used successfully in the treatment of human maladies, including liver dysfunction, betaine, shown to protect against the early stages of alcoholic liver injury as well as being a SAM generator, may become a promising therapeutic agent and a possible alternative to expensive SAM in the treatment of liver disease and other human maladies.

Methionine inhibits developmental aggregation of Myxococcus xanthus by blocking the biosynthesis of S-adenosyl methionine

Previous studies showed that high concentrations of methionine (> 1 mM) inhibited aggregation and fruiting body formation in Myxococcus xanthus (E. Rosenberg, D. Filer, D. Zafriti, and S. H. Kindler, J. Bacteriol. 115: 29-34, 1973, and J. M. Campos and D. R. Zusman, Proc. Natl. Acad. Sci. USA 72:518-522, 1975). However, the mechanism for the inhibition was unclear. In this study, we found that high levels of methionine inhibited the biosynthesis of S-adenosylmethionine (SAM) and that reduced intracellular levels of SAM are correlated with defective chemotactic movements and reduced developmental gene expression. In addition, we found that methionine analogs and high concentrations of amino acids which are known to affect SAM synthesis in other bacteria, such as threonine, lysine, and isoleucine, also caused reduced cellular levels of SAM and blocked fruiting body formation in M. xanthus. These results indicate that SAM is required for development of M. xanthus and the inhibitory effect of methionine on development results, at least in part, from its blocking of the biosynthesis of SAM.

Mutations affecting the biosynthesis of S-adenosylmethionine cause reduction of DNA methylation in Neurospora crassa.

A temperature-sensitive methionine auxotroph of Neurospora crassa was found in a collection of conditional mutants and shown to be deficient in DNA methylation when grown under semipermissive conditions. The defective gene was identified as met-3, which encodes cystathionine-gamma-synthase. We explored the possibility that the methylation defect results from deficiency of S-adenosylmethionine (SAM), the presumptive methyl group donor. Methionine starvation of mutants from each of nine complementation groups in the methionine (met) pathway (met-1, met-2, met-3, met-5, met-6, met-8, met-9, met-10 and for) resulted in decreased DNA methylation while amino acid starvation, per se, did not. In most of the strains, including wild-type, intracellular SAM peaked during rapid growth (12-18 h after inoculation), whereas DNA methylation continued to increase. In met mutants starved for methionine, SAM levels were most reduced (3-11-fold) during rapid growth while the greatest reduction in DNA methylation levels occurred later. Addition of 3 mM methionine to cultures of met or cysteine-requiring (cys) mutants resulted in 5-28-fold increases in SAM, compared with wild-type, at a time when DNA methylation was reduced approximately 40%, suggesting that the decreased methylation during rapid growth in Neurospora is not due to limiting SAM. DNA methylation continued to increase in a cys-3 mutant that had stopped growing due to methionine starvation, suggesting that methylation is not obligatorily coupled to DNA replication in Neurospora.

MTX does not affect enhanced biosynthesis and metabolism of S-adenosylmethionine in testosterone-induced hypertrophic mouse kidney.

In the testosterone-induced hypertrophic female mouse kidney model we recently found that one of the anabolic effects of testosterone, apart from its profound induction of ornithine decarboxylase, which results in the stimulation of putrescine biosynthesis, is the enhancement of synthesis and levels of S-adenosylmethionine (AdoMet), and an increase in cellular methylation intensity1. Synthesis of renal AdoMet may be considered to be a folate-dependent process, because of the involvement of 5-methyltetrahydrofolate in the methylation of homocysteine to methionine. Blocking by MTX of tetrahydrofolate regeneration from dihydrofolate causes cellular depletion of reduced folates2, among them 5-methyltetrahydrofolate3,4. In addition, MTX also decreases levels of AdoMet5,6, choline and betaine6 in various rat tissues, and causes an increase in urinary wasting of endogenous folates in the rat kidney7.

A mutant of Escherichia coli temperature sensitive in the biosynthesis of S-adenosylmethionine

A mutant of Escherichia coli temperature sensitive in the biosynthesis of S-adenosylmethionine

A mutant ofEscherichia colitemperature sensitive in the biosynthesis ofS-adenosylmethionine

S-Adenosylmethionine (SAM) is synthesized in vitro by SAM synthetase, which is coded for by the metK gene of Escherichia coli. Since E. coli cells are impermeable to SAM it has been impossible to obtain mutants which require SAM for growth. The finding that SAM is required for regulating methionine biosynthesis was used to select metK mutants which have the phenotype of resistance to methionine analogues. These mutants show reduced SAM synthesis in vitro, and a lower level of intracellular SAM. However, as all these mutants can grow, without added SAM, the block in SAM synthesis must be incomplete. To try to block SAM synthesis more completely we combined temperature-sensitive metK mutation with a block in the synthesis of cystathionine (metA), a precursor that supports only slow growth because of slow entry. At the non-permissive temperature for metK these double mutants could grow on methionine but not on cystathionine, in correlation with the greatly reduced intracellular level of SAM. This result indicates that it is possible to reduced intracellular SAM concentrations to levels which are low enough to prevent growth of E. coli. Moreover, since the mutation responsible for the reduction in SAM level is in the metK gene, it is suggested that SAM synthetase is the enzyme which catalyses SAM biosynthesis in E. coli cells.

A kinetic isotope effect study and transition state analysis of the S-adenosylmethionine synthetase reaction.

The biosynthesis of S-adenosylmethionine occurs in a unique enzymatic reaction in which the synthesis of the sulfonium center results from displacement of the entire polyphosphate chain from MgATP. The mechanism of S-adenosylmethionine synthetase (ATP:L-methionine s-adenosyltransferase) from Escherichia coli has been characterized by kinetic isotope effect and substrate trapping measurements. Replacement of 12C by 14C at the 5' carbon of ATP yields a primary Vmax/Km isotope effect (12C/14C) of 1.128 +/- 0.003 in the absence of added monovalent cation activator (K+). At saturating K+ concentrations (10 mM) the primary isotope effect diminishes slightly to 1.108 +/- 0.003, indicating that the step in the mechanism involving bond breaking at the 5' carbon of MgATP has a small commitment to catalysis at conditions near Vmax. No alpha-secondary 3H isotope effect from [5'-3H]ATP was detected, (1H/3H) = 1.000 +/- 0.002, even in the absence of KCl. There was no significant primary sulfur isotope effect from [35S]methionine at KCl concentrations from 0 to 10 mM. Substitution of the methyl group of methionine with tritium yielded a beta-secondary isotope effect (CH3/C3H3) = 1.009 +/- 0.008 independent of KCl concentration. The reaction of selenomethionine and [5'-14C]ATP gave a primary isotope effect of 1.097 +/- 0.006, independent of KCl concentration. Substrate trapping experiments demonstrated that the step in the mechanism involving bond making to sulfur of methionine does not have a significant commitment to catalysis at 0.25 mM KCl, therefore intrinsic isotope effects were observed. Substrate trapping experiments indicated that the step involving bond breaking at carbon 5' of MgATP has a 10% commitment to catalysis at 0.25 mM KCl. The isotope effects are interpreted in terms of an Sn2-like transition state structure in which bonding of the C5' is symmetric with respect to the departing tripolyphosphate group and the incoming sulfur of methionine. With selenomethionine as substrate an earlier transition state is implicated.

Growth inhibition by methionine analog inhibitors of S-adenosylmethionine biosynthesis in the absence of polyamine depletion

Four methionine analog inhibitors of methionine adenosyltransferase, the enzyme which catalyzes S-adenosylmethionine biosynthesis, were tested in cultured L1210 cells for their effects on cell growth, leucine incorporation, S-adenosylmethionine (AdoMet) formation and polyamine biosynthesis. The IC 50 values were as follows: selenomethionine, 0.13 mM; L-2-amino-4-methoxy- cis -but-3-enoic acid (L- cis -AMB), 0.4 mM; cycloleucine, 5 mM and 2-aminobicyclo[2.1.1]hexane-2-carboxylic acid, 5 mM. At IC 50 levels, the analogs significantly reduced AdoMet pools by ∼50% while not similarly affecting leucine incorporation or polyamine biosynthesis. In combination with inhibitors of polyamine biosynthesis, growth inhibition was greatly increased with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase, but only slightly increased with α-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Overall, the data indicate that the methionine analogs, and particularly L- cis -AMB, seem to inhibit cell growth by interference with AdoMet biosynthesis. Since polyamine biosynthesis is not affected, the antiproliferative effect may be mediated through perturbations of certain transmethylation reactions.

Effect of inhibitors of methylation on early and late interferon synthesis in bovine kidney cell cultures.

In virus infected bovine kidney cell cultures mainly late interferon is produced starting at about 4 hr after infection. Poly rI:poly rC induced cells as well as interferon pretreated virus infected cells produce early interferon starting immediately after induction. In infected cells the proportion of early interferon increases with time of interferon pretreatment, while late interferon is decreasing. Production of late interferon is selectively inhibited by cycloleucine, an inhibitor of S-adenosylmethionine (SAM) biosynthesis, whereas early interferon synthesis is not affected by the drug. Likewise, late interferon production is reduced much stronger than early interferon production by a combination of adenosine, L-homocysteine thiolactone, and erythro-9[3-(2-hydroxynonyl)]-adenine (EHNA) inducing in cells an accumulation of S-adenosylhomocysteine which inhibits SAM mediated methylation reactions. Inhibition of late interferon synthesis by cycloleucine is time and dose dependent and partially reversible. Cycloheximide equally blocks both early and late interferon production. Inhibition of incorporation of methyl groups into cellular RNA by the methylation inhibitors used is demonstrated by labeling with [methyl-3H] methionine and 3H-uridine. The results indicate that the synthesis of functional mRNA for early and late interferon is differentially sensitive to inhibition of methylation. The data suggest that, if early and late interferon is coded by the same structural gene, two different pathways are available for the cell to synthesize one species of mRNA.

Stimulation of yeast ascospore germination and outgrowth by S-adenosylmethionine

The supplementation of S-adenosylmethionine (SAM) to germination medium stimulated the accumulation of [14C]uracil from the medium into germinating cells, as well as its incorporation into ribonucleic acid during germination and outgrowth of ascospores of Saccharomyces cerevisiae. In addition to uracil, the accumulation of leucine, cytosine, serine, and methionine was also stimulated by the extracellular addition of this sulfonium compound. The SAM-stimulatory effect was dose dependent; half-maximal stimulation was observed at about 50 muM. The effect exerted by SAM supplementation appeared to be specific for SAM and for germination and outgrowth. In the absence of SAM biosynthesis (in the presence of cycloleucine), spores were inhibited in their ability to accumulate label, whereas the supplementation of SAM completely reversed the cycloleucine-induced inhibition of accumulation. In addition to accumulation and incorporation, the kinetics of bud formation during outgrowth were also stimulated by exogenous SAM. The stimulation of budding by SAM was amplified in an ethionine-resistant strain. These observations suggest that SAM may be essential for the initiation of cell division during the breaking of spore dormancy.

Vesicular stomatitis virus mRNA methylation in Vivo: Effect of cycloleucine, an inhibitor of S-adenosylmethionine biosynthesis, on viral transcription and translation

Cycloleucine is a potent and specific inihibitor of S-adenosylmethionine biosynthesis and subsequent RNA methylations in Chinese hamster ovary (CHO) cells. When added in the course of an infection of CHO cells with vesicular stomatitis virus (VSV), cycloleucine does not significantly modify the rate of viral RNA and protein synthesis or the production of mature particles. Under the same conditions cycloleucine will reduce by 83% the methyolation of newly synthesized polyadenylated 11–16 S viral RNA. The mode of action of cycloleucine and cycloheximide are clearly distinguishable under these conditions. In contrast when cells are treated with cycloleucine, immediately after virus adsorption, the effect on viral RNA synthesis is very similar to that of cycloheximide. That is, a 90% inhibition in the production of viral messenger RNAs is observed along with a complete disappearance of newly synthesized 38 S RNA species, suggesting that secondary transcription is inhibited. Viral protein synthesis and virus production are similarly inhibited during this period. The undermethylated mRNAs of VSV synthesized in the presence of cycloleucine are 80% undertranslated as compared to mRNAs synthesized in untreated cells. Our results suggest that mRNA methylation in VSV is required for translation in vivo. The mode of action of cycloleucine during the infectious process is discussed.

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

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