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.     

Inhibition of translation of mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase by polyamines

The effect of spermidine and spermine on the translation of the mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase was studied using a reticulocyte lysate system and specific antisera to precipitate these proteins. It was found that the synthesis of these key enzymes in the biosynthesis of polyamines was much more strongly inhibited by the addition of polyamines than was either total protein synthesis or the synthesis of albumin. Translation of the mRNA for S-adenosylmethionine decarboxylase was maximal in a lysate which had been substantially freed from polyamines by gel filtration. Addition of 80 microM spermine had no significant effect on total protein synthesis and stimulated albumin synthesis but reduced the production of S-adenosylmethionine decarboxylase by 76%. Similarly, addition of 0.8 mM spermidine reduced the synthesis of S-adenosylmethionine decarboxylase by 82% while albumin and total protein synthesis were similar to that found in the gel-filtered lysate. Translation of ornithine decarboxylase mRNA was greater in the gel-filtered lysate than in the control lysate but synthesis of ornithine decarboxylase was stimulated slightly by low concentrations of polyamines and was maximal at 0.2 mM spermidine or 20 microM spermine. Higher concentrations were strongly inhibitory with a 70% reduction occurring at 0.8 mM spermidine or 150 microM spermine. Further experiments in which both polyamines were added together confirmed that the synthesis of ornithine and S-adenosylmethionine decarboxylases were much more sensitive to inhibition by polyamines than protein synthesis as a whole. These results indicate that an important part of the regulation of polyamine biosynthesis by polyamines is due to a direct inhibitory effect of the polyamines on the translation of mRNA for these biosynthetic enzymes.     

Cloning, mapping and mutational analysis of the S-adenosylmethionine decarboxylase gene in Drosophila melanogaster

S-adenosylmethionine decarboxylase is a key enzyme in the synthesis of polyamines. These small cationic molecules are required for growth and development in all organisms. A wealth of biological processes, including synthesis of DNA and protein and condensation of chromatin, involve polyamines. Inhibition of polyamine synthesis has been proposed for treatment of cancer but this requires more knowledge about the in vivo function of polyamines. We report here the cloning of the S-adenosylmethionine decarboxylase gene from Drosophila melanogaster and the analysis of corresponding mutants. The mutant phenotypes are similar to those previously described for ribosomal protein genes (Minutes) and rRNA genes (bobbed?). This work elucidates the in vivo consequences of impaired polyamine synthesis with respect to the development of a whole animal.     

The effect of polyamines on ethylene synthesis during normal and pollination-induced senescence of Petunia hybrida L. flowers

Senescence of Petunia hybrida L. flowers is accompanied by a climacteric pattern in ethylene production and a rapid decline in the levels of putrescine and spermidine during the preclimacteric phase. The decrease in spermidine is caused by the decline in the availability of putrescine which is initially synthesized from L-arginine via agmatine and N-carbamoylputrescine. Inhibition of putrescine and polyamine synthesis resulted in a rapid drop in the levels of putrescine and spermidine without resulting in a concomitant increase in ethylene production. These results indicate that polyamine synthesis is not involved in the control of ethylene synthesis through its effect on the availability of S-adenosylmethionine, and is confirmed by the results obtained with pollinated flowers. Treatment with polyamines may stimulate or suppress ethylene production in the corolla, depending on the concentrations applied. In unpollinated flowers the onset of the climacteric rise in ethylene production was accelerated after treatment with polyamines. However, in pollinated flowers this process was delayed as a result of treatment with low concentrations of polyamines. The effects of exogenous polyamines on ethylene production in both pollinated and unpollinated flowers indicate that ethylene synthesis in these flowers is not regulated by a feedback control mechanism. Although polyamines do not play a key role in the control of ethylene production during the early stages of senescence through their effect on the availability of S-adenosylmethionine, it appears that they play an important role in some of the other processes involved in senescence.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - MGBG methylglyoxal bis-(guanylhydrazone) - SAM S-adenosylmethionine     

Cloning, mapping and mutational analysis of the S-adenosylmethionine decarboxylase gene in Drosophila melanogaster

S-adenosylmethionine decarboxylase is a key enzyme in the synthesis of polyamines. These small cationic molecules are required for growth and development in all organisms. A wealth of biological processes, including synthesis of DNA and protein and condensation of chromatin, involve polyamines. Inhibition of polyamine synthesis has been proposed for treatment of cancer but this requires more knowledge about the in vivo function of polyamines. We report here the cloning of the S-adenosylmethionine decarboxylase gene from Drosophila melanogaster and the analysis of corresponding mutants. The mutant phenotypes are similar to those previously described for ribosomal protein genes (Minutes) and rRNA genes (bobbed ). This work elucidates the in vivo consequences of impaired polyamine synthesis with respect to the development of a whole animal. Received: 16 May 1997 / Accepted: 25 July 1997     

Endogenous polyamines are intimately associated with highly condensed chromatin in vivo. A fluorescence cytochemical and immunocytochemical study of spermine and spermidine during the cell cycle and in reactivated nuclei

Polyamines are low molecular weight aliphatic polycations essential for cell proliferation and differentiation. By immunocytochemistry, as well as by two independent fluorescence cytochemical methods, we show that polyamines are associated with highly condensed chromatin in nucleated erythrocytes and in metaphase and anaphase chromosomes. In other cells, polyamines mainly occur in cytoplasm. The association between polyamines and DNA in condensed chromatin is so close that DNase treatment is necessary for making polyamines available for reaction with antibodies. Studies of chick/HeLa cell heterokaryons reveal that polyamines disappear from the chick erythrocyte nuclei concomitantly with DNA decondensation and initiation of RNA synthesis. Our data strongly suggest that polyamines are important for chromatin condensation in vivo.     

Function of S-adenosylmethionine in germinating yeast ascospores.

Germination and outgrowth of ascospores of Saccharomyces cerevisiae 4579 require both methionine and adenine, whereas leucine is only required for outgrowth. The methionine requirement may be satisfied by S-adenosylmethionine, but this sulfonium compound will not substitute for adenine. Between 30 and 70 min of protein synthesis is initially required for the completion of germination in strain 4579. The inhibition of S-adenosylmethionine synthetase by trifluoromethionine prevents both germination and protein synthesis. During the initial stages of germination, the S-adenosylmethionine synthetase, S-adenosylmethionine decarboxylase, and transfer ribonucleic acid methyltransferases increased significantly, indicating that polyamines and/or the methylation of transfer ribonucleic acid are required for the initiation of germination.     

In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from the S-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism.     

Active transport and metabolic characteristics of polyamines in the rat lens

Putrescine, spermidine and spermine were transported into the rat lens against a concentration gradient. This process appeared to be energy-dependent and involved a carrier system different from those for amino acids. Competition experiments suggested that the three polyamines were transported by the same system or very similar systems. Incorporated spermine was converted to spermidine and putrescine, and spermidine was converted to putrescine. In contrast, the conversion of putrescine to spermidine and spermine, or the conversion of spermidine to spermine was not observed. Furthermore, ornithine was not utilized for the synthesis of putrescine. These metabolic characteristics of the polyamines in the rat lens were correlated with the extremely low activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase. Other enzymes of polyamine metabolisms, however, were relatively active. In conclusion, the lens has a very low ability for the de novo synthesis of polyamines. The polyamines in the lens are considered to be supplied form the surrounding intraocular fluid by an active transport system specific for polyamines.     

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.     

Cellular systems for the study of the biosynthesis of polyamines and ethylene,as well as of virus multiplication

Leaves of Chinese cabbage from healthy plants or from those infected with turnip yellow mosaic virus yield protoplasts which convert methionine to protein, S-adenosylmethionine, decarboxylated S-adenosylmethionine, spermidine, spermine and 1-aminocyclopropane-1-carboxylate. The enzyme spermidine synthase is entirely cytosolic and has been purified extensively. An inhibitor of this enzyme, dicyclohexylamine, blocks spermidine synthesis in intact protoplasts, and in so doing stimulates spermine synthesis. Aminoethoxyvinylglycine blocks the conversion of S-adenosylmethionine to 1-aminocyclopropane-1-carboxylate, the precursor to ethylene, in protoplasts. This inhibitor markedly stimulates the synthesis of both spermidine and spermine. Essentially all the protoplasts obtained from new leaves of plants infected 7 days earlier are infected. On incubation, such protoplasts convert exogenous methionine to viral protein and viral spermidine whose specific radioactivity is twice that of total cell spermidine. Exogeneous spermidine is also converted to cell putrescine and viral spermidine and spermine. Normal and virus-infected cells are being studied for their content of phenolic acid amides of the polyamines.     

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.     

Putrescine does not support the migration and growth of IEC-6 cells

The migration of IEC-6 cells is inhibited when the cells are depleted of polyamines by inhibiting ornithine decarboxylase with alpha-difluoromethylornithine (DFMO). Exogenous putrescine, spermidine, and spermine completely restore cell migration inhibited by DFMO. Because polyamines are interconverted during their synthesis and catabolism, the specific role of individual polyamines in intestinal cell migration, as well as growth, remains unclear. In this study, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone)(DEGBG), to block the synthesis of spermidine and spermine from putrescine. We found that exogenous putrescine does not restore migration and growth of IEC-6 cells treated with DFMO plus DEGBG, whereas exogenous spermine does. In addition, the normal distribution of actin filaments required for migration, which is disrupted in polyamine-deficient cells, could be achieved by adding spermine but not putrescine along with DFMO and DEGBG. These results indicate that putrescine, by itself, is not essential for migration and growth, but that it is effective because it is converted into spermidine and/or spermine.     

Methionine utilization in long-term human lymphoid cell lines

Long-term lymphoid cell lines (LTL) cultured under normal conditions use methionine primarily for protein synthesis, although a significant proportion is converted to S-adenosylmethionine (SAM) and used for synthesis of the polyamines, spermidine, and spermine. When LTL are cultured under conditions of high cell density, there is an initial phase of rapid protein synthesis and accumulation of SAM as found under normal culture conditions, but this soon ceases. Polyamine synthesis is small under these conditions, despite the presence of relatively large amounts of SAM.     

Intracellular polyamine patterns during encystment of Physarum flavicomum

In Physarum flavicomum Berk., growing amoebae convert to dormant cysts under conditions of nutrient imbalance. Exogenous adenine inhibits the process and the cells produce an elevated intracellular concentration of S-adenosylmethionine. Evidence indicates that the increased level of S-adenosylmethionine is responsible for the disruption of the normal developmental process. One of the biological functions of S-adenosylmethionine is in polyamine synthesis and it is known that a well-controlled intracellular concentration of polyamines is essential for normal cell growth and differentiation. In this study, high-performance liquid chromatography was used to determine the intracellular polyamine patterns in growing cells, adenine-treated and normal encysting cells, and dormant cysts. Putrescine and spermidine were the most abundant polyamines found in the cells; growing cells had the highest level, adenine-treated cells had a 1.5 to 2.0 times higher level than normal encysting cells, while cysts had the lowest (only 3 and 12% of that of growing cells). Cadaverine and N1-acetylspermidine were found in all the cells and their levels decreased during encystment. Acetylputrescine was found in growing cells only and acetylspermine was found in all cells except cysts. Acetylcadaverine, N8-acetylspermidine, 1,3-diaminopropane, and spermine were not detected in any of the cells.     

Transgenic mouse models for studies of the role of polyamines in normal,hypertrophic and neoplastic growth

Transgenic mice expressing proteins altering polyamine levels in a tissue-specific manner have considerable promise for evaluation of the roles of polyamines in normal, hypertrophic and neoplastic growth. This short review summarizes the available transgenic models. Mice with large increases in ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase or antizyme, a protein regulating polyamine synthesis by reducing polyamine transport and ODC in the heart, have been produced using constructs in which the protein is expressed from the alpha -myosin heavy-chain promoter. These mice are useful in studies of the role of polyamines in hypertrophic growth. Expression from keratin promoters has been used to target increased synthesis of ODC, spermidine/spermine-N(1)-acetyltransferase (SSAT) and antizyme in the skin. Such expression of ODC leads to an increased sensitivity to chemical and UV carcinogenesis. Expression of antizyme inhibits carcinogenesis in skin and forestomach. Expression of SSAT increases the incidence of skin papillomas and their progression to carcinomas in response to a two-stage carcinogenesis protocol. These results establish the importance of polyamines in carcinogenesis and neoplastic growth and these transgenic mice will be valuable experimental tools to evaluate the importance of polyamines in mediating responses to oncogenes and studies of cancer chemoprevention.     

Phosphatidylethanolamine methyltransferase activity in chick liver microsomes

The synthesis of phosphatidylcholine from phosphatidylethanolamine is carried out by chick liver microsomes (Gallus domesticus). Different concentrations of PE, NPE and NNPE were used as exogenous substrates. Saturation of the S-adenosylmethionine has been found for the three different reactions with or without exogenous substrate. Kinetic parameters have been determined for this enzyme system in chick liver microsomes. The three methyl reactions had a similar pH profile with an optimum at pH = 8. Divalent ions such as Ca2+ or Mg2+ did not stimulate the enzyme activity. The results suggest that the synthesis of phosphatidylcholine from phosphatidylethanolamine by chick liver microsomes exhibits a kinetic pattern with different aspects than that described for other animal or human preparations.     

Polyamine auxotrophs of Saccharomyces cerevisiae.

Strains of yeast have been constructed that are unable to synthesize ornithine and are thereby deficient in polyamine biosynthesis. These strains were used to develop a protocol for isolation of mutants blocked directly in polyamine synthesis. There were seven mutants isolated that lack ornithine decarboxylase activity; these strains exhibited greatly decreased pool levels of putrescine, spermidine, and spermine when grown in the absence of polyamines. Three of the mutants lack S-adenosylmethionine decarboxylase activity; polyamine limitation of a representative mutant resulted in an accumulation of putrescine and a decrease in spermidine and spermine. When the mutants were cultured in the absence of polyamines, a continuously declining growth rate was observed.     

Peach (Prunus persica) fruit ripening: aminoethoxyvinylglycine (AVG) and exogenous polyamines affect ethylene emission and flesh firmness

The effect of various concentrations of aminoethoxyvinylglycine (AVG; 0.32 and 1.28 m M ), an ethylene biosynthesis inhibitor, and of the polyamines putrescine (10 m M ), spermidine (0.1, 1 and 5 m M ) and spermine (2 m M ) on peach ( Prunus persica L. Batsch cv. Redhaven) fruit ripening was evaluated under field conditions. Treatments were performed 19 (polyamines) and 8 (AVG) days before harvest. Fruit growth (diameter, fresh and dry weight), flesh firmness, soluble solids content and ethylene emission were determined on treated and untreated (controls) fruits. Moreover, endogenous polyamine content and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) activity were determined to check for a possible competition between polyamines and ethylene for their common precursor S-adenosylmethionine (SAM). Both treatments strongly inhibited ethylene emission and delayed flesh softening. On a biochemical level, AVG and exogenous polyamines both reduced the free-to-conjugate ratio of endogenous polyamines, and transiently altered SAMDC activity. The possible use of these compounds to control fruit ripening is discussed also in the light of their rejuvenating effect on peach fruits.     

Regulated translation termination at the upstream open reading frame in s-adenosylmethionine decarboxylase mRNA.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a cis-acting element that confers feedback control by cellular polyamines on translation of this message. Recent studies demonstrated that elevated polyamines inhibit synthesis of the peptide encoded by the uORF by stabilizing a ribosome paused in the vicinity of the termination codon. These studies suggested that polyamines act at the termination step of uORF translation. In this paper, we demonstrate that elevated polyamines stabilize an intermediate in the termination process, the complete nascent peptide linked to the tRNA that decodes the final codon. The peptidyl-tRNA molecule is found associated with the ribosome fraction, and decay of this molecule correlated with release of the paused ribosome from the message. Furthermore, the stability of this complex is influenced by the same parameters that influence regulation by the uORF in vivo, namely the concentration of polyamines and the sequence of the uORF-encoded peptide. These results suggest that the regulated step in uORF translation is after formation of the peptidyl-tRNA molecule but before hydrolysis of the peptidyl-tRNA bond. This regulation may involve an interaction between the peptide, polyamines, and a target in the translational apparatus.