Biochemical analysis of the role of transmethylation in the methionine dependence of tumor cells.

The effects on methionine metabolism of the substitution of homocysteine for methionine in vitro were investigated in normal and tumor cell lines differing in their ability to utilize homocysteine for growth. The major finding of this study was that methionine-independent (Met-Indep) cell lines had much lower basal transmethylation rates than methionine-dependent (Met-Dep) cell lines. This was particularly evident in the parent SP1 cell line and its Met-Indep revertant, SP1-R. SP1-R compensated for a lack of methionine by reducing both its transmethylation and growth rates. An analysis of other potential differences in methionine metabolism between Met-Dep and Met-Indep cell lines failed to demonstrate any consistent abnormalities in all but the absolutely Met-Dep MDAY-D2 cell line. Thus, protein, S-adenosylmethionine, and polyamine synthesis were the same in Met-Dep and Met-Indep cell lines. These results indicate that the major regulatory step in determining the Met-Dep phenotype is an inherent increase in the rate of transmethylation reactions. Cell lines with high basal transmethylation rates cannot compensate for a relative deficiency of methionine and either cease growing (MDAY-D2) or generate revertants (SP1-R) for which the basal rate of transmethylation is considerably reduced.

Formation of S-adenosylhomocysteine in the heart. II: A sensitive index for regional myocardial underperfusion.

Rate of accumulation of myocardial S-adenosylhomocysteine (SAH) was used in an open-chest dog preparation as an index of free cytosolic adenosine levels. Following 30 minutes of coronary artery ligation and infusion of L-homocysteine thiolactone (10 mumol/kg/min i.v.) SAH levels increased from 1.3 (control) to 3.3 nmoles/g in the nonischemic and to values over 100 nmoles/g in the ischemic region. Compared with regional myocardial blood flow the enhanced rate of SAH accumulation was strictly confined to the ischemic area. As long as blood flow was 0.6-1.2 ml/min/g, SAH levels remained unchanged. However, they steeply increased when regional myocardial blood flow decreased below 60% of control. Tissue levels of adenine nucleotides, adenosine, and lactate were not significantly affected in the flow range of 0.4-0.6 ml/min/g but rate of SAH accumulation was enhanced by 400%. In the nonischemic myocardium, SAH accumulation was 60% higher in the subendocardium than in the subepicardium. Decreasing coronary perfusion pressure from 110 to 60, 45, and 35 mm Hg was associated with an exponential increase in coronary venous adenosine release only when perfusion pressure was below 60 mm Hg. Transmural mapping of SAH revealed that at 110 mm Hg SAH was homogeneously distributed, while at a perfusion pressure of 60 mm Hg SAH accumulation was enhanced only in the subendocardial layers. Decreasing perfusion pressure further to 40 and 30 mm Hg not only enhanced subendocardial SAH levels to 120 and 170 nmoles/g, respectively, but also considerably steepened the transmural gradient of SAH. SAH-hydrolase exhibited a broad pH-optimum and its activity in different parts of ventricular myocardium was identical. Our findings provide evidence that 1) measurement of SAH accumulation is a sensitive metabolic index for the assessment of regional myocardial ischemia, 2) significant formation of SAH occurs only when regional myocardial blood flow is less than 0.6 ml/min/g, and 3) transmural SAH gradient, a measure of free cytosolic adenosine, and coronary venous adenosine release significantly increase only when the autoregulatory reserve is exhausted.

Formation of S-adenosylhomocysteine in the heart. I: An index of free intracellular adenosine.

To assess the concentration of free intracellular adenosine in the heart the kinetic properties of cytosolic S-adenosylhomocysteine (SAH) hydrolase were utilized at elevated levels of L-homocysteine (adenosine + L-homocysteine in equilibrium with SAH + H2O). Global hypoxia was induced in the isolated perfused guinea pig heart by graded reduction of perfusion medium PO2 in the presence of saturating concentrations of homocysteine (0.2-1.0 mM). Reduction of PO2 from 660 to 165 mm Hg increased the steady-state concentration of total tissue adenosine from 2.0 +/- 0.2 to 2.8 +/- 0.2 nmoles/g, while the rate of SAH formation increased linearly from 0.22 +/- 0.03 to 2.50 +/- 0.13 nmoles/min/g. When adenosine was exogenously applied at a concentration of 100 microM together with homocysteine (1 mM), SAH accumulation rates were much greater: 23.34 +/- 3.31 and 42.11 +/- 1.73 nmoles/min/g with normoxic (95% O2) and hypoxic (30% O2) perfusion, respectively. The apparent Km and Vmax values for SAH-hydrolase in vivo were estimated to be 20 microM and 59 nmoles/min/g wet wt, respectively. Since the relation between SAH formation and adenosine in the physiological concentration range is linear, the measured rate of SAH accumulation during normoxia and hypoxia permitted the calculation of the free intracellular adenosine level, which was 0.061 nmoles/g (0.08 microM) in the normoxic heart. With hypoxia (PO2 165 mm Hg), this value increased to 1.57 nmoles/g (2.0 microM). Free intracellular adenosine closely correlated with the hypoxia-induced changes in coronary flow. The data reveal that measurement of the rate of SAH accumulation during homocysteine infusion can be used for sensitive assessment of free intracellular adenosine levels. Assuming that the intracellular adenosine concentration equals that in the interstitial space, the results furthermore indicate that the degree of intracellular adenosine formation during hypoxic perfusion is quantitatively sufficient to account for most of the observed increases in coronary flow.

S-adenosylmethionine: studies on chemical and enzymatic synthesis.

Several methods for the chemical and enzymatic synthesis of (-)-S-adenosylmethionine (AdoMet) are described and compared. Studies on the effects of solvents, pH, methylating reagents, and KI on the coupling of sodium homocysteine thiolate and 5'-chloro-5'-deoxyadenosine led to an improved procedure for the synthesis of (+/-)-AdoMet. The use of trimethylsulfonium iodide as a methylating agent under acidic conditions results in a higher content of the desired (-)-epimer than does the use of CH3I. The enzymatic synthesis of (-)-AdoMet using AdoMet synthetase from an over-producing strain of Escherichia coli is demonstrated and the effect of product inhibition on preparative-scale synthesis is illustrated. A new HPLC technique for separation of the epimeric mixture of AdoMet, which allows small-scale preparation of optically pure AdoMet from the enzyme product, has been developed. With this HPLC technique, evidence that (-)-AdoMet is the sole epimer formed by the enzyme has been shown.

3-Deazaneplanocin A: a new inhibitor of S-adenosylhomocysteine synthesis and its effects in human colon carcinoma cells.

The mechanism of action of the cyclopentenyl analogue of 3-deazaadenosine (3-deazaneplanocin A or c3Nep) was investigated in the human colon carcinoma cell line HT-29. Upon exposure of cells for 24 hr to 3-deazaneplanocin A (c3Nep), neplanocin A (Nep) or 3-deazaaristeromycin (c3Ari), significant toxicity was noted only for Nep, wherein an 87% reduction in viability was produced at a 100 microM concentration. c3Nep and c3Ari at 100 microM reduced viability by 34 and 21%, respectively. Intracellular levels of S-adenosylhomocysteine (AdoHcy) were elevated by a 24-hr exposure to 100 microM c3Nep, Nep and c3Ari and were 120, 75 and 25 pmoles/10(6) cells respectively. Only Nep was metabolized to an S-adenosylmethionine-like metabolite, and its formation was dose-related to its cytotoxicity. The t1/2 for the disappearance of elevated levels of AdoHcy following drug removal was 1.6 to 2.5 hr for all drugs. rRNA and tRNA methylation was inhibited significantly by Nep, but c3Nep and c3Ari inhibited tRNA methylation but not rRNA methylation to a lesser degree. These results demonstrate that c3Nep is a potent inhibitor of AdoHcy synthesis with a low degree of cytotoxicity.

Inhibition of equine S-adenohomocysteine hydrolase by 2'-deoxyadenosine.

2'-Deoxyadenosine and 9-beta-D-arabinofuranosyladenine (ARA) are apparent suicide inhibitors for equine S-adenosylhomocysteine hydrolase. In initial velocity studies of the synthetic reaction converting adenosine and homocysteine to S-adenosylhomocysteine, adenine, adenosine 5'-triphosphate, and 9-beta-D-arabinofuranosyladenine were found to be competitive inhibitors with Kis of 3.8 microM, 1.1 mM, and 30 microM, respectively. In contrast, linear mixed inhibition was observed for 2'-deoxyadenosine, indicating that 2'-deoxyadenosine must bind in more than one fashion to the enzyme.

Adenosine metabolism in a rat hippocampal slice preparation: incorporation into S-adenosylhomocysteine.

The incorporation of [14C]adenosine into various metabolites was studied in a hippocampal slice preparation in order to assess the extent of adenosine metabolism via synthesis of S-adenosylhomocysteine, a potent inhibitor of transmethylation reactions. Highest incorporation of 14C occurred into nucleotides, with only a few percent being recovered in inosine + hypoxanthine, S-adenosylhomocysteine, and the free adenosine pool. Labeling of S-adenosylhomocysteine did not significantly increase with higher concentrations of added adenosine despite greater accumulation of free [14C]adenosine in the tissue. Addition of L-homocysteine significantly increased the labelling of S-adenosylhomocysteine. The results indicate that S-adenosylhomocysteine synthesis is a minor pathway of adenosine metabolism in brain tissue under steady-state conditions. Further, changes in adenosine concentration, without a concomitant change in L-homocysteine availability, are unlikely to lead to a significant accumulation of S-adenosylhomocysteine. S-Adenosylhomocysteine is therefore not likely to play a significant role in mediating the biological effects of adenosine in the CNS via inhibition of transmethylations.

Biosynthesis of S-N6-methyladenosylhomocysteine, an inhibitor of RNA methyltransferases.

This paper demonstrates that N6-methyladenosine (6-methylaminopurine ribonucleoside) will condense in vitro with homocysteine to form S-N6-methyladenosylhomocysteine in a reaction catalyzed by mouse liver S-adenosylhomocysteine hydrolase. Injection of mice with N6-methyladenosine is followed by accumulation of S-N6-methyladenosylhomocysteine in the liver. Studies from other laboratories have shown that S-N6-methyladenosylhomocysteine is nearly as potent an RNA methyltransferase inhibitor as S-adenosylhomocysteine. This indicates that administration of N6-methyladenosine may be a general method for blocking in vivo RNA methylation in studies to determine the role of methylation in RNA processing and translational function.

S-adenosylmethionine metabolism and its relation to polyamine synthesis in rat liver. Effect of nutritional state, adrenal function, some drugs and partial hepatectomy.

S-Adenosylmethionine metabolism and its relation to the synthesis and accumulation of polyamines was studied in rat liver under various nutritional conditions, in adrenalectomized or partially hepatectomized animals and after treatment with cortisol, thioacetamide or methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidine)dinitrilo]diguanidine}. Starvation for 2 days only slightly affected S-adenosylmethionine metabolism. The ratio of spermidine/spermine decreased markedly, but the concentration of total polyamines did not change significantly. The activity of S-adenosylmethionine decarboxylase initially decreased and then increased during prolonged starvation. This increase was dependent on intact adrenals. Re-feeding of starved animals caused a rapid but transient stimulation of polyamine synthesis and also increased the concentrations of S-adenosylmethionine and S-adenosylhomocysteine. Similarly, cortisol treatment enhanced the synthesis of polyamines, S-adenosylmethionine and S-adenosylhomocysteine. Feeding with a methionine-deficient diet for 7-14 days profoundly increased the concentration of spermidine, whereas the concentrations of total polyamines and of S-adenosylmethionine showed no significant changes. The results show that nutritional state and adrenal function play a significant role in the regulation of hepatic metabolism of S-adenosylmethionine and polyamines. They further indicate that under a variety of physiological and experimental conditions the concentrations of S-adenosylmethionine and of total polyamines remain fairly constant and that changes in polyamine metabolism are not primarily connected with changes in the accumulation of S-adenosylmethionine or S-adenosylhomocysteine.

The relation between purine metabolism and flavinogenesis in Eremothecium ashbyii. The identification of S-adenosylmethionine and S-adenosylhomocysteine accumulated in non-growing cells of E. ashbyii.

When adenine was added to the non-growing cell medium of Eremothecium ashbyii, riboflavin production of the mold was increasingly inhibited with increasing concentration of adenine. Under these conditions, a cationic compound was accumulated in the mycelia. The compound was isolated from the mycelia and highly purified. The purified compound was proved to be S-adenosylhomocysteine through IR analysis. In the control experiment, or in the addition of other purines which stimulated riboflavin production of the mold, another cationic compound was accumulated in the non-growing cells. The compound was largely accumulated in the presence of both adenine and methionine, isolated from the mycelia and purified. The purified compound was concluded to be S-adenosylmethionine through IR and NMR analyses. The significance of these compounds on the riboflavin biosynthetic pathway was argued under "Discussion".

A new method for the assay of tissue. S-adenosylhomocysteine and S-adenosylmethione. Effect of pyridoxine deficiency on the metabolism of S-adenosylhomocysteine, S-adenosylmethionine and polyamines in rat liver.

The hepatic synthesis and accumulation of S-adenosylhomocysteine, S-adenosylmethionine and polyamines were studied in normal and vitamin B-6-deficient male albino rats. A method involving a single chromatography on a phosphocellulose column was developed for the determination of S-adenosylhomocysteine and S-adenosylmethionine from tissue samples. Feeding the rat with pyridoxine-deficient diet for 3 or 6 weeks resulted in a four- to five-fold increase in the concentration of S-adenosylhomocysteine, whereas that of S-adenosylmethionine was only slighly elevated. The concentration of putrescine was decreased to half, that of spermidine was somewhat decreased and that of spermine remained fairly constant. The activities of L-ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, L-methionine adenosyltransferase and S-adenosyl-L-homocysteine hydrolase were moderately increased. S-Adenosylmethionine decarboxylase showed no requirement for pyridoxal 5'-phosphate. The major effect of pyridoxine deficiency of S-adenosylmethionine metabolism seems to be a block in the utilization of S-adenosylhomocysteine, resulting in the accumulation of this metabolite to a concentration that may inhibit biological methylation reactions.