Selenite toxicity,depletion of liver S-adenosylmethionine,and inactivation of methionine adenosyltransferase

The possibility that dimethyl selenide production depletes liver S-adenosylmethionine was explored as a biochemical basis for selenite toxicity. Toxic doses of selenite (25 nmol/ g body weight) were found to rapidly decrease mouse liver S-adenosylmethionine and increase S-adenosylhomocysteine, indicative of an increased rate of transmethylation. However, S-adenosylmethionine levels remained depressed beyond the time when dimethyl selenide synthesis ceased, suggesting that selenite inactivated methionine adenosyltransferase. This was found to be the case in vivo by measuring the effect of graded doses of selenite on the conversion of the methionine analog, ethionine, to S-adenosylethionine. In vitro studies also indicated inactivation of this enzyme by selenite. Liver homogenates from mice injected with 25 nmol of selenite/g, as above, were found to have less than 50% of the methionine adenosyltransferase activity of saline-injected controls.     

Effects of nitrous oxide-induced inactivation of cobalamin on methionine and S-asenosylmethionine metabilism in the rat

Inhalation of nitrous oxide oxidises cobalamin and, in turn, inactivates methionine synthetase which forms methionine from homocysteine and which requires cob[I]alamin as a co-factor. This study was planned to determine the effect of virtual cessation of methionine synthesis via a cobalamn-dependeent pathway, on tissue levels of methionine, S-adenosylmethionine and on related enzymes. The level of methionine in liver fell initially after exposure to N₂O but was restored to pre-N₂O levels after 6 days despite continuing N₂O exposure. Brain methionine fell within 12 h of N₂O exposure but the fall was not significant. The restoration of methionine levels is accompanied by an increase in activity of betaine homoysteine methyltransferase in liver but this enzyme was not detected in brain. The activity of methionine synthetase remained very low in both liver and brain as long as N₂O inhalation was continued. There was an initial rise in liver S-adenosyl-methionine levels followed by a steady fall to 40% of its initial level after 11 days of N₂O exposure. However, there was no change in the level of S-adenosylmethionine in brain during this period. The data indicate that either brain meets its requirement by increased methionine uptake from plasma or that there are alternate pathways in brain for methionine synthesis other than those requiring a cobalamin coenzyme.     

Intracellular localization of aspartate kinase and the enzymes of threonine and methionine biosynthesis in green leaves

The intracellular localization of several aspartate pathway enzymes has been studied in pea (Pisum sativum cv Feltham First) and barley (Hordeum vulgare cv Julia) leaves. Protoplast lysates were fractionated by differential or sucrose density gradient centrifugation, in media optimized for each enzyme. The results show that aspartate kinase, homoserine kinase, threonine synthase, and cystathionine γ-synthase are confined to the chloroplast. Cystathionine β-lyase appears to be present in several fractions, though more than 50% of the total activity is associated with the chloroplasts. In contrast, neither methionine synthase nor methionine adenosyl-transferase were significantly associated with chloroplasts, and only a small proportion of the methionine synthase was associated with the mitochondrial fraction. Methionine adenosyltransferase, and hence S-adenosylmethionine synthesis, is not found in any organelle fraction. The conclusion is that whereas threonine, like lysine, is synthesized only in the chloroplast, the last step in methionine biosynthesis occurs largely in the cytoplasm.     

Interactions of Methionine and Selenomethionine with Methionine Adenosyltransferase and Ethylene-generating Systems

Since selenomethionine appears to be a better precursor of ethylene in senescing flower tissue of Ipomoea tricolor and in indole acetic acid-treated pea stem sections than is methionine (Konze JR, N Schilling, H Kende 1978 Plant Physiol 62: 397-401), we compared the effectiveness of selenomethionine and methionine to participate in reactions which may be connected to ethylene biosynthesis. Evidence is presented that selenomethionine is also a better substrate of methionine adenosyltransferase (ATP: methionine S-adenosyltransferase, EC 2.5.1.6) from I. tricolor, the V_max for selenomethionine being twice as high as that for methionine. The affinity of the enzyme is higher for methionine than for selenomethionine, however. Methionine added to flower tissue together with selenomethionine inhibits the enhancement of ethylene synthesis by the seleno analog. Likewise, methionine reduces the high, selenomethionine-dependent reaction rates of methionine adenosyltransferase from I. tricolor flower tissue. On the other hand, selenomethionine is less effective as an ethylene precursor than is methionine in model systems involving oxidation by free radicals. It was concluded that activation of methionine by methionine adenosyltransferase and formation of S-adenosylmethionine are more likely to be involved in ethylene biosynthesis than is oxidation of methionine by free radicals.     

Methionine biosynthesis in isolated Pisum sativum mitochondria

Homocysteine-dependent transmethylases utilizing 5-methyltetrahydropteroylglutamic acid and S-adenosylmethionine as methyl donors have been examined using ammonium sulphate fractions prepared from isolated mitochondria of pea cotyledons. Substantial levels of a 5-rnethyltetrahydropteroylglutamate transmethylase were detected, the catalytic properties of this enzyme being found similar to those of a previously reported enzyme present in cotyledon extracts. The mitochondrial 5-CH₃-H₄PteGlu transmethylase had an apparent K_m of 25 μM for the methyl donor, was saturated with homocysteine at 1 mM and was inhibited 50% by l-methionine at 2.5 mM. At similar concentrations of methyl donor the mitochondrial S-adenosylmethionine methyltransferase was not saturated. Mitochondrial preparations were found capable of synthesizing substantial amounts of S-adenosylmethionine but lacked ability to form S-methylmethionine. Significant levels of β-cystathionase, cystathionine-γ-synthase, l-homoserine transacetylase and l-homoserine transsuccinylase were detected in the isolated mitochondria. The activity of the enzymes of homocysteine biosynthesis was not affected by l-methionine in vitro. It is concluded that pea mitochondria have ability to catalyze the synthesis of methionine de novo.     

Assay for S-adenosylmethionine: methionine methyltransferase

A quantitative assay for S-adenosylmethionine: methionine methyltransferase in phosphate buffer extracts has been developed. This enzyme catalyzes the biosynthesis of S-methylmethionine from methionine and S-adenosylmethionine. The radioactively labeled product, S-methylmethionine, is first separated from the radioactively labeled substrate, l-methionine, by means of ion-exchange chromatography. Once separated thusly, the amount present can then be directly determined by the use of a liquid scintillation spectrometer.     

Comparison of the effects of buthioninesulfoximine and phorone on the metabolism of sulfur-containing amino acids in rat liver

Alterations in hepatic transsulfuration reactions were determined in rats treated with a glutathione-depleting agent. A dose of l-buthionine-(S, R)-sulfoximine decreased hepatic methionine, cysteine, S-adenosylmethionine, and glutathione levels rapidly. Methionine adenosyltransferase and γ-glutamylcysteine lygase activities were decreased transiently, but significantly. The activity of cysteine dioxygenase was increased, resulting in an elevation of hypotaurine and taurine concentrations. Administration of phorone reduced hepatic glutathione and cysteine similarly, but S-adenosylmethionine concentrations were elevated for as long as 72 h. Hepatic methionine adenosyltransferase, cystathionine β-synthase, cystathionine γ-lyase, and γ-glutamylcysteine lygase activities were all increased but cysteine dioxygenase activity and taurine generation were markedly depressed. The results show that a decrease in hepatic GSH induces profound changes in sulfur amino acid metabolomics, which would subsequently influence various cellular processes. It is suggested that the change in hepatic levels of sulfur-containing substances and its physiological significance should be considered when a glutathione-depleting agent is utilized in biological experiments.     

Over-expression of cystathionine γ-synthase in Arabidopsis thaliana leads to increased levels of methionine and S-methylmethionine

Cystathionine γ-synthase (CGS, EC 4.2.99.9), the first committed enzyme in methionine biosynthesis, was over-expressed in Arabidopsis thaliana by introducing in the genome of this plant the coding sequence of the Arabidopsis enzyme under the control of the cauliflower mosaic virus 35S promoter. In order to target the recombinant protein to the chloroplast, the transgene included the sequence encoding the N-terminal transit peptide of Arabidopsis CGS. CGS activity and polypeptide were increased several fold in these plants. There was a markedly increased level of soluble methionine in the leaves of the transformed plants, up to 15-fold, indicating that CGS is a rate-limiting enzyme in this metabolic pathway. In addition, the transformed plants strongly over-accumulated S-methylmethionine, but not S-adenosylmethionine, in agreement with the view that S-methylmethionine corresponds to a storage form of labile methyl groups in plants and/or plays a role in preventing S-adenosylmethionine accumulation. The same strategy was used to increase the level of cystathionine β-lyase (CBL, EC 4.4.1.8), the second committed enzyme in methionine biosynthesis, in transformed A. thaliana. Despite an increase in both CBL activity and polypeptide in transformed Arabidopsis plants over-expressing Arabidopsis CBL, there was very little change in the contents of soluble methionine and S-methylmethionine, suggesting strongly that CBL is not rate limiting in the methionine biosynthetic pathway.     

Transfer RNA methyltransferase and glycine N-methyltransferase activity during Rana pipiens development.

Changes in the activity of the tRNA methyltransferases have been found in all differentiating systems studied. Activity was examined in extracts of Rana pipiens embryos and in larval and adult liver by in vitro assay using S-adenosyl-l-[methyl-¹⁴C]methionine as the methyl donor. Specific activities of tRNA methyltransferases decreased, beginning with the time of feeding, when using high concentrations of the crude liver enzyme. A new methyltransferase activity, glycine N-methyltransferase, appeared at the time of feeding. Apparently, the glycine methyltransferase is active before the onset of any of the characteristic metamorphic changes of other liver enzymes. Using partially purified enzyme from adult liver, the K_m of glycine methyltransferase for S-adenosylmethionine is 0.3 mM and the K_i for S-adenosylhomocysteine, a competitive inhibitor, is 0.08 mM.     

Effect of adenosine metabolites on methyltransferase reactions in isolated rat livers

Adenosine is rapidly metabolized by isolated rat livers. The major products found in the perfusate were inosine and uric acid while hypoxanthine could also be detected. S-Adenosylhomocysteine was also excreted when the liver was perfused with both adenosine and L-homocysteine. A considerable portion of the added adenosine was salvaged via the adenosine kinase reaction. The specific radioactivity of the resultant AMP reached 75–80% of the added [8-¹⁴C]adenosine within 90 min. When the liver was perfused with adenosine alone, hydrolysis of S-adenosyllhomosysteine, via S-adenosylhomocysteine hydrolase, appeared to be blocked resulting in the accumulation of this compound. As the intracellular level of S-adenosylhomocysteine increased, the rates of various methyltransferase reactions were reduced, resulting in elevated levels of intracellular S-adenosylmethionine. When the liver was perfused with normal plasma levels of methionine the S-adenosylmethionine : S-adenosylhomocysteine ratio was 5.3 and the half-life of the methyl groups was 32 min. Upon further addition of adenosien the S-adenosylmethionine : S-adenosylhomocysteine ratio shifted to 1.7 and the half-life of the methyl groups to 103 min. In the presence of adenosine and L-homocysteine such inordinate amounts of S-adenosylhomocysteine accumulated in the cell that methylation reactions were completely inhibited. Although adenine has been found to be a product of the S-adenosylhomocysteine hydrolase only trace quantities of this compound were detectable in the tissue after perfusing the liver with high concentrations of adenosine for 90 min.     

The S-Methylmethionine Cycle in Lemna paucicostata

The metabolism of S-methylmethionine has been studied in cultures of plants of Lemna paucicostata and of cells of carrot (Daucus carota) and soybean (Glycine max). In each system, radiolabeled S-methylmethionine was rapidly formed from labeled l-methionine, consistent with the action of S-adenosyl-l-methionine:methionine S-methyltransferase, an enzyme which was demonstrated during these studies in Lemna homogenates. In Lemna plants and carrot cells radiolabel disappeared rapidly from S-methylmethionine during chase incubations in nonradioactive media. The results of pulse-chase experiments with Lemna strongly suggest that administered radiolabeled S-methylmethionine is metabolized initially to soluble methionine, then to the variety of compounds formed from soluble methionine. An enzyme catalyzing the transfer of a methyl group from S-methylmethionine to homocysteine to form methionine was demonstrated in homogenates of Lemna. The net result of these reactions, together with the hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine, is to convert S-adenosylmethionine to methionine and adenosine. A physiological advantage is postulated for this sequence in that it provides the plant with a means of sustaining the pool of soluble methionine even when overshoot occurs in the conversion of soluble methionine to S-adenosylmethionine. The facts that the pool of soluble methionine is normally very small relative to the flux into S-adenosylmethionine and that the demand for the latter compound may change very markedly under different growth conditions make it plausible that such overshoot may occur unless the rate of synthesis of S-adenosylmethionine is regulated with exquisite precision. The metabolic cost of this apparent safeguard is the consumption of ATP. This S-methylmethionine cycle may well function in plants other than Lemna, but further substantiating evidence is neeeded.     

Alterations in the metabolomics of sulfur-containing substances in rat kidney by betaine

Earlier studies have shown that betaine administration may modulate the metabolism of sulfur amino acids in the liver. In this study, we determined the changes in the metabolomics of sulfur-containing substances induced by betaine in the kidney, the other major organ actively involved in the transsulfuration reactions. Male rats received betaine (1 %) in drinking water for 2 weeks before killing. Betaine intake did not affect betaine–homocysteine methyltransferase activity or its protein expression in the renal tissue. Expression of methionine synthase was also unchanged. However, methionine levels were increased significantly both in plasma and kidney. Renal methionine adenosyltransferase activity and S-adenosylmethionine concentrations were increased, but there were no changes in S-adenosylhomocysteine, homocysteine, cysteine levels or cystathionine β-synthase expression. γ-Glutamylcysteine synthetase expression or glutathione levels were not altered, but cysteine dioxygenase and taurine levels were decreased significantly. In contrast, betaine administration induced cysteine sulfinate decarboxylase and its metabolic product, hypotaurine. These results indicate that the metabolomics of sulfur-containing substances in the kidney is altered extensively by betaine, although the renal capacity for methionine synthesis is unresponsive to this substance unlike that of the liver. It is suggested that the increased methionine availability due to an enhancement of its uptake from plasma may account for the alterations in the metabolomics of sulfur-containing substances in the kidney. Further studies need to be conducted to clarify the physiological/pharmacological significance of these findings.     

Vitamin B12-dependent Methionine Biosynthesis and Its Metabolic Role in Corynebacterium simplex ATCC 6946, a Vitamin B12-producing and Hydrocarbon-utilizable Bacterium

A vitamin B₁₂-dependent N⁵-methyltetrahydrofoIate-homocysteine methyltransferase was found in cell-free extracts of Corynebacterium simplex ATCC 6946 grown aerobically in a medium containing hydrocarbon as a sole carbon source and the enzyme was partially purified. Absolute requirements for S-adenosylmethionine and an appropriate reducing system were observed for the transmethylation from N⁵-methyltetrahydrofolate. The same preparation catalyzed also the formation of methionine from homocysteine and methyl-B₁₂ under both aerobic and anaerobic conditions. The concentration of cobalt ion in the growth medium had a pronounced effect on the intracellular vitamin B₁₂ level and the activity of the vitamin-dependent methionine-synthesizing system in the bacterium. The relationship between the methionine synthesis and the methyl branched-chain fatty acid formation was discussed.     

Synthesis of methylated ethanolamine moieties: regulation by choline in soybean and carrot

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[³H₃C]methionine, l-[¹⁴CH₃]methionine, or [1,2-¹⁴C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.     

Regulation of methionine biosythesis in Neurospora crassa.

The regulation of serine hydroxymethyltransferase, methylenetetrahydrofolate reductase, and methyltetrahydropteroylpolyglutamate:homocysteine methyltransferase was investigated in Neurospora crassa. Adding choline to the medium decreased the specific activity of these enzymes. Methionine potentiated the choline effect, but, when added alone, was without effect. Neither choline, methionine, nor S-adenosylmethionine appears to be the immediate corepressor of synthesis of these enzymes.Several nonallelic mutants were examined for the enzymes of methionine methyl group synthesis. The formate-requiring mutant for lacks serine hydroxymethyltransferase. The methionine-requiring mutants me-1 and me-8 lack, respectively, the reductase and the methyltransferase. The methionine-requiring mutants me-1, me-6 (folate polyglutamate synthetase deficient) and me-8 were found to have significantly higher serine hydroxymethyltransferase specific activities than did the wild-type strain.     

The geometry of the thioester group and its implications for the chemistry of acyl coenzyme A

L-929 cell surface membranes were incubated with S-adenosyl-l-[methyl-³H]-methionine and found to contain phosphatidylethanolamine: S-adenosylmethionine N-methyltransferase (phosphatidylethanolamine N-methyltransferase) activity. The enzyme or combination of enzymes responsible for this activity methylated endogenous phosphatidylethanolamine and its methylated derivatives to yield phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine. Maximum enzyme activity was expressed at pH 6.9, the reaction was not dependent on the presence of divalent cations, and exogenously added phospholipids did not stimulate the rate of reaction. Phospholipid methylation was inhibited by S-adenosyl-l-homocysteine and by local anaesthetic drugs such as chlorpromazine and tetracaine which partition into the lipid bilayer. Control experiments demonstrated that the surface membrane-associated methyltransferase activity was not due to contamination of surface membrane preparations with intracellular membranes. Surface membranes were found to have higher specific methyltransferase activities than whole L-cell homogenates or endoplasmic reticulum-enriched microsomes. The low rate of methyltransferase function expressed in vitro (approximately 1 pmol/min · mg protein) suggests that phospholipid methylation is not a major metabolic source of surface membrane phosphatidylcholine.     

Regulation of Auxin-induced Ethylene Production in Mung Bean Hypocotyls: Role of 1-Aminocyclopropane-1-Carboxylic Acid

Ethylene production in mung bean hypocotyls was greatly increased by treatment with 1-aminocyclopropane-1-carboxylic acid (ACC), which was utilized as the ethylene precursor. Unlike auxin-stimulated ethylene production, ACC-dependent ethylene production was not inhibited by aminoethoxyvinylglycine, which is known to inhibit the conversion of S-adenosylmethionine to ACC. While the conversion of methionine to ethylene requires induction by auxin, the conversion of methionine to S-adenosylmethionine and the conversion of ACC to ethylene do not. It is proposed that the conversion of S-adenosylmethionine to ACC is the rate-limiting step in the biosynthesis of ethylene, and that auxin stimulates ethylene production by inducing the synthesis of the enzyme involved in this reaction.     

Guanidoacetate methyltransferase from rat liver: Purification,properties, and evidence for the involvement of sulfhydryl groups for activity

Guanidoacetate methyltransferase (EC 2.1.1.2) has been purified about 800-fold from rat liver. The purified preparation shows a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme is estimated to be 25,000 and 26,000 by Sephadex gel molecular-exclusion chromatography and by electrophoresis in polyacrylamide gradient gel, respectively. The sodium dodecyl sulfate-denatured enzyme also has a molecular weight of 26,000; thus, the enzyme is a monomeric protein. Guanidoacetate methyltransferase as isolated is catalytically inactive, but is readily reactivated by incubation with a thiol. The reactivated enzyme, which contains 3 mol of sulfhydryl groups/mol of enzyme, is again inactivated by oxidized glutathione. This inactivation is accompanied by the disappearance of two sulfhydryl residues. The relationship between the loss of enzyme activity and the number of residues disappeared indicates that the integrity of these sulfhydryl residues is critical for activity. The oxidized enzyme fails to bind the substrate S-adenosylmethionine as evidenced by the equilibrium dialysis study. Alkylation of the nonoxidizable sulfhydryl by N-ethylmaleimide shows that this residue is also essential for activity. UV absorption, fluorescence, and CD spectra show no difference between the reduced and oxidized enzymes, but the former is more susceptible to proteolytic attack by trypsin. The enzyme has an isoelectric pH of 5.3, and is most active at pH 9.0. From the CD spectrum, an α helix content of 15% is calculated. The K_m values for guanidoacetate and S-adenosylmethionine are 97.5 and 6.73 μm, respectively, at pH 8.0 and 37 °C.     

5-Methyl-Tetrahydrofolate and the S-Adenosylmethionine Cycle in C57BL/6J Mouse Tissues: Gender Differences and Effects of Arylamine N-Acetyltransferase-1 Deletion

Folate catabolism involves cleavage of the C⁹-N¹⁰ bond to form p-aminobenzoylgluamate (PABG) and pterin. PABG is then acetylated by human arylamine N-acetyltransferase 1 (NAT1) before excretion in the urine. Mice null for the murine NAT1 homolog (Nat2) show several phenotypes consistent with altered folate homeostasis. However, the exact role of Nat2 in the folate pathway in vivo has not been reported. Here, we examined the effects of Nat2 deletion in male and female mice on the tissue levels of 5-methyl-tetrahydrofolate and the methionine-S-adenosylmethionine cycle. We found significant gender differences in hepatic and renal homocysteine, S-adenosylmethionine and methionine levels consistent with a more active methionine-S-adenosylmethionine cycle in female tissues. In addition, methionine levels were significantly higher in female liver and kidney. PABG was higher in female liver tissue but lower in kidney compared to male tissues. In addition, qPCR of mRNA extracted from liver tissue suggested a significantly lower level of Nat2 expression in female animals. Deletion of Nat2 affected liver 5- methyl-tetrahydrofolate in female mice but had little effect on other components of the methionine-S-adenosylmethionine cycle. No N-acetyl-PABG was observed in any tissues in Nat2 null mice, consistent with the role of Nat2 in PABG acetylation. Surprisingly, tissue PABG levels were similar between wild type and Nat2 null mice. These results show that Nat2 is not required to maintain tissue PABG homeostasis in vivo under normal conditions.     

Coupling of the polyamine and iron metabolism pathways in the regulation of proliferation: Mechanistic links to alterations in key polyamine biosynthetic and catabolic enzymes

Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N¹-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced ³H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the “reprogramming” of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation.