The effect of methionine, ethylene and polyamine catabolic intermediates on polyamine accumulation in detached soybean leaves

In the present study we determined the effects of methionine, intermediates of polyamine catabolic pathways and inhibitors of either ethylene biosynthetic or polyamine catabolic pathways on polyamine accumulation in soybean leaves. Inhibitors to SAM decarboxylase and spermidine synthase, methylglyloxal-bis-(guanylhy-drazone) and cyclohexylamine, respectively, suggest that methionine may provide aminopropyl groups for the synthesis of polyamine via S-adenosylmethionine (SAM). Results from experiments that utilized a combination of compounds which altered either ethylene or polyamine biosynthesis, namely, aminoethoxyvinyl glycine, CoSO₄, 2,5-norbornadiene, and CuSO₄, suggest the two pathways compete for a common precursor. However, exogenous addition of ethylene (via ethephon treatments) had little or no effect on polyamine biosynthesis. Likewise, polyamine treatments had little or no effect on ethylene biosynthesis. These data suggest that there are few or no inhibitory effects from the end products of one pathway on the synthesis of the other. Data from leaves treated with metabolic intermediates in the catabolic pathway of polyamines and inhibitors of enzymes in the catabolic pathway, i.e. aminoguanidine, hydroxyethyldrazine and gabaculine, suggest that the observed increases in polyamine titers were not due to decreased catabolism of the polyamines. One catabolic intermediate, γ-aminobutyric acid (GABA), elevated putrescine, spermidine and spermine by 12-, 1.4-, and 2-fold, respectively, Ethylene levels decreased (25%) in GABA-treated leaves. This small decrease in ethylene could not account for such large increase in putrescine titers. Further analysis demonstrated that the GABA-mediated polyamine accumulation was inhibited by difluoromethylarginine, an inhibitor of arginine decarboxylase, but not by difluoromethylornithine, an inhibitor of ornithine decarboxylase. These data suggest that GABA directly or indirectly affects the biosynthesis of polyamines via arginine decarboxylase.     

百合小鳞茎离体发生过程中内源多胺水平和多胺氧化酶活性的变化

以百合小鳞茎离体发生为实验系统,研究了鳞片(外植体)内多胺和多胺氧化酶活性的变化与小鳞茎发生之间的相关性。随着细胞脱分化、愈伤组织形成、小鳞茎发生等过程,内源腐胺、亚精胺和精胺的含量均失上升后下降,变化曲线为“钟形”,其含量达最高峰的时间为肉眼可见小鳞茎出现之前。多胺氧化酶活性在小鳞茎发生初期逐渐下降,6d后开始上升,并在小鳞茎突起时达最大值。由于内源多胺水平和多胺氧化酶活性的变化与小鳞茎发生有密切关系,可作为这一形态发生过程的生化指示。     

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.     

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     

Polyamine Synthesis and Interconversion by the Microsporidian Encephalitozoon cuniculi

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.     

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.     

Polyamine levels in relation to growth and NaCl concentration in normal and habituated sugarbeet callus cultures

Abstract. The concentrations of putrescine, spermidine and spermine, the only polyamines detectable in normal and habituated calli of Beta vulgaris L. ssp. altissima , were much higher in the habituated callus than the normal callus, irrespective of experimental conditions. These results suggest that, in normal (tolerant to NaCl) and habituated (sensitive to NaCl) calli, there exists a competition for the common precursor of ethylene and polyamine biosynthesis viz. S-adenosylmethionine. A disequilibrium favouring the synthesis of putrescine and spermidine in the habituated callus might be linked to structural deterioration of the cell membrane following extended culture or severe osmotic stress (68 mol m⁻¹ NaCl). The maintenance of membrane integrity by the normal callus coincides with ethylene production at the expense of polyamine synthesis. In contrast to the habituated callus, the salinity tolerance of the normal callus is accompanied by the accumulation of proline under hypersaline conditions (274mol m⁻³). The important osmoregulatory role played by quaternary ammonium compounds in the-aerial parts of Chenopodiaceae, especially the sugarbeet, is not observed in the calli, these compounds being found in very low concentrations in saline conditions.     

Polyamine biosynthesis in the developing rabbit palate

Levels of putrescine, spermidine, and spermine and their biosynthetic enzymes, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) were measured in the developing rabbit palate between day 14 and day 18 of gestation. DNA, RNA, and protein synthesis were also measured during this time period to determine if a correlation exists between polyamine biogenesis and macromolecular synthesis. ODC activity was found to be twice as high on day 14 as on the succeeding days of gestation, while SAMDC activity did not change significantly. Levels of putrescine and spermine were higher on day 14 by 22% and 30%, respectively, than levels on day 18. Spermidine concentration did not change. DNA synthesis remained relatively constant between days 14 and 18 of gestation, suggesting that there is no peak in cell proliferation during this period. RNA synthesis was elevated significantly on day 14 and protein synthesis was significantly higher on both days 14 and 16. This data indicates that there is no correlation between polyamine synthesis and cell proliferation during this period of palatal development, but polyamines could play a regulatory role in RNA and/or protein synthesis.     

Polyamine metabolism in enterocytes isolated from newborn pigs.

In the pig, the growth of intestinal mucosa is very intense after birth. Since the polyamines are key elements affecting cell proliferation and differentiation, the present work was undertaken in order to know whether this hypertrophy is associated with an adaptation of polyamine metabolism. Villus enterocytes isolated from pig immediately after birth or 2 days later were found to contain similar amounts of putrescine, spermidine and spermine, i.e., 0.23; 0.41 and 1.24 nmol/10(6) cells, respectively. At birth, despite a relatively high ODC activity, putrescine synthesis from 1 mM L-arginine or 2 mM L-glutamine was very low in isolated enterocytes (6.4 +/- 3.8 pmol/10(6) cells per 30 min), while spermidine and spermine production were not detectable. This could be explained by a very low L-ornithine generation from both amino acids and to an inhibitory effect of polyamines on ODC activity. Two days later, polyamine synthesis from L-arginine remained undetectable despite a higher L-ornithine generation. This was concomitant with a dramatic fall in ODC activity. At both stages, enterocytes were able to take up polyamines from the extracellular medium in a temperature-dependent manner. It is concluded that de-novo synthesis of polyamines from L-arginine or L-glutamine does not play a significant role in the control of polyamine content of pig enterocytes during the postnatal period. In contrast, polyamine uptake by enterocytes would contribute to maintain a steady-state polyamine content during this period.     

Polyamine metabolism in some helminth parasites

Polyamine levels of some helminth parasites were analyzed by reverse phase HPLC of benzoyl derivatives. Setaria cervi, Acanthocheilonema viteae, Hymenolepis nana, H. diminuta, and Ascaridia galli contained higher levels of spermine than spermidine while in Ancylostoma ceylanicum and Nippostrongylus brasiliensis the spermidine levels were higher than spermine; putrescine was either absent or present in minor quantities. The enzymes of polyamine biosynthesis viz., ornithine decarboxylase, S-adenosyl methionine (SAM)-decarboxylase, and arginine decarboxylase were present in very low to negligible amounts in all the parasites examined. A. ceylanicum exhibited high activity of ornithine amino transferase (OAT) and catalyzed appreciable decarboxylation of ornithine. The ornithine decarboxylating activity of A. ceylanicum was localized in the particulate fraction containing mitochondria, not inhibited by alpha-difluoromethyl ornithine, the specific inhibitor of ornithine decarboxylase (ODC), but inhibited in the presence of glutamate, suggesting the involvement of mitochondrial OAT rather than a true ODC in ornithine decarboxylation in this parasite. Significant activity of polyamine oxidase was also detected in helminth parasites. The absence of polyamine biosynthesizing enzymes in helminth parasites suggests their dependence on hosts for uptake and interconversion of polyamines, providing a potential target for chemotherapy.     

Polyamine transport,accumulation, and release in brain

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.     

Met对渗透胁迫下小麦幼苗内源多胺含量和乙烯产生的影响

渗透胁迫下,小麦幼苗内源多胺含量和乙烯产生均明显增加,再用0．4mmoL甲硫氨酸掺人处理后,乙烯释出加速,精胺含量进一步增多,亚精胺含量变化不大,腐胺的含量几乎减少到胁迫前的水平。可见,渗透胁迫下,甲硫氨酸既可以在Met循环中以甲硫氨酸与腐胺联合生成亚精胺,进而再与亚精胺联合生成精胺,又可以S-腺苷甲硫氨酸分解生成5’-甲硫基腺苷和氨基环丙烷羧酸,最后由氨基环丙烷羧酸加氧生成乙烯。     

采后梨果实皮层和髓中EFE活性、ACC和多胺含量变化及其与乙烯生成的关系

菊水梨皮层和髓的乙烯合成酶（ＥＦＥ）活性和氨基环丙烷羧酸（ＡＣＣ）含量及其果实乙烯释放量均分别高于二十世纪梨。在同一品种果实的皮层和髓中,ＡＣＣ含量无明显差异,但髓的ＥＦＥ活性明显高于皮层。梨果实内含有丁二胺、亚精胶和精胺３种内源多胺,而以丁二胺和亚精胺含量为最高,精胺含量虽低但较稳定。果实在乙烯释放高峰出现前,其皮层和髓中的丁二胺和亚精胺含量变化有明显差异。采后梨果实的乙烯生成与果实内ＥＦＥ活性,ＡＣＣ含量、多胺含量的变化有密切关系,而乙烯生成的差异不仅表现在品种间,而且在果实的皮层和髓之间也存在差异,梨果实的髓似乎对整个果实的乙烯生成有更重要的影响。     

Changes in Polyamine Contents Development of the Frog, Microhyla ornata

Putrescine, spermidine and spermine levels were measured during development, metamorphosis and adult life of the frog, Microhyla ornata . Development of Microhyla was accompanied by high fluctuating levels of putrescine and spermidine with low and steady levels of spermine. Putrescine was the major polyamine during development from egg to mature tadpole. During metamorphosis both putrescine and spermidine decreased significantly; but the decrease in putrescine content was more rapid than that of spermidine. Thus, in the freshly metamorphosed frog, the concentration of spermidine exceeded that of putrescine. In most of the adult tissues also spermidine concentration was higher than putrescine and spermine. While the free form of putrescine and spermidine increased during early development of the fertilized egg to tadpole, the levels of protein conjugated polyamines decreased. In the free form, putrescine was the major polyamine while in the protein conjugated form spermidine concentration was higher than putrescine and spermine. Thus polyamine pattern is different in early development, during metamorphosis and in differentiated adult tissues of this frog. ∞-Difluoromethylornithine treatment at early blastula stage did not interfere with the normal development of Microhyla embryos.     

Polyamine synthesis from proline in the developing porcine placenta

Polyamines (putrescine, spermidine, and spermine) are essential for placental growth and angiogenesis. However, little is known about polyamine synthesis in the porcine placenta during conceptus development. The present study was conducted to test the hypothesis that arginine and proline are the major sources of ornithine for placental polyamine production in pigs. Placentae, amniotic fluid, and allantoic fluid were obtained from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, and 110 of the 114-day gestation (n = 6 per day). Placentae as well as amniotic and allantoic fluids were analyzed for arginase, proline oxidase, ornithine aminotransferase (OAT), ornithine decarboxylase (ODC), proline transport, concentrations of amino acids and polyamines, and polyamine synthesis using established radiochemical and chromatographic methods. Neither arginase activity nor conversion of arginine into polyamines was detected in the porcine placenta. In contrast, both proline and ornithine were converted into putrescine, spermidine, and spermine in placental tissue throughout pregnancy. The activities of proline oxidase, OAT, and ODC as well as proline transport, polyamine synthesis from proline, and polyamine concentrations increased markedly between Days 20 and 40 of gestation, declined between Days 40 and 90 of gestation, and remained at the reduced level through Day 110 of gestation. Proline oxidase and OAT, but not arginase, were present in allantoic and amniotic fluids for the production of ornithine (the immediate substrate for polyamine synthesis). The activities of these two enzymes as well as the concentrations of ornithine and total polyamines in fetal fluids were highest at Day 40 but lowest at Days 20, 90, and 110 of gestation. These results indicate that proline is the major amino acid for polyamine synthesis in the porcine placenta and that the activity of this synthetic pathway is maximal during early pregnancy, when placental growth is most rapid. Our novel findings provide a new base of information for future studies to define the role of proline in fetoplacental growth and development.     

Polyamine biosynthesis and inhibition in Trichomonas vaginalis

Polyomines - particularly putrescine, spermidine and spermine - are ubiquitous components of eukaryote and most prokaryote cells, and are essential for optimal cell proliferation. But since routes of polyamine synthesis may differ, for example between parasites and their hosts, selective inhibition of polyamine metabolism offers an attractive target for chemotherapy - as already shown with the success of difluoromethylomithine (DFMO) as an inhibitor of polyamine synthesis in African trypanosomes. Parasitology Today has featured a series of articles reviewing research on polyamine metabolism of various parasites (eg. vol. 3, pp 190-192, pp 312-315; vol. 4, pp 18-20) and here, Nigel Yorlett discusses these metabolic aspects of Trichomonas vaginalis (Fig. 1)-a common parasite of the urogenital tract.     

Inhibition of the ethylene response by 1-MCP in tomato suggests that polyamines are not involved in delaying ripening, but may moderate the rate of ripening or over-ripening

Ethylene initiates the ripening and senescence of climacteric fruit, whereas polyamines have been considered as senescence inhibitors. Ethylene and polyamine biosynthetic pathways share S-adenosylmethionine as a common intermediate. The effects of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, on ethylene and polyamine metabolism and associated gene expression was investigated during ripening of the model climacteric fruit, tomato (Solanum lycopersicum L.), to determine whether its effect could be via polyamines as well as through a direct effect on ethylene. 1-MCP delayed ripening for 8 d compared with control fruit, similarly delaying ethylene production and the expression of 1-aminocyclopropane-1-carboxylic acid (ACC)-synthase and some ethylene receptor genes, but not that of ACC oxidase. The expression of ethylene receptor genes returned as ripening was reinitiated. Free putrescine contents remained low while ripening was inhibited by 1-MCP, but increased when the fruit started to ripen; bound putrescine contents were lower. The activity of the putrescine biosynthetic enzyme, arginine decarboxylase, was higher in 1-MCP-treated fruit. Activity of S-adenosylmethionine-decarboxylase peaked at the same time as putrescine levels in control and treated fruit. Gene expression for arginine decarboxylase peaked early in non-treated fruit and coincident with the delayed peak in putrescine in treated fruit. A coincident peak in the gene expression for arginase, S-adenosylmethionine-decarboxylase, and spermidine and spermine synthases was also seen in treated fruit. No effect of treatment on ornithine decarboxylase activity was detected. Polyamines are thus not directly associated with a delay in tomato fruit ripening, but may prolong the fully-ripe stage before the fruit tissues undergo senescence.     

Polyamine content in Arabidopsis thaliana (L.) Heynh during recovery after low and high temperature treatments

Comparative studies on the effect of temperature treatment on the endogenous polyamine content in wild type and the ethylene insensitive mutant eti5 of Arabidopsis thaliana (L.) Heynh were performed. The levels of free and conjugated putrescine, spermidine and spermine were measured in rosette leaves of 38-day-old plants subjected to low and high temperature for 24 h in darkness. Data for fractions measured in treated wild type plants during recovery suggest that alterations in polyamine levels may be a consequence of the conversion of the supernatant-bound into free form and vice versa, while in treated eti5 plants de novo synthesis of spermidine and spermine could not be excluded. It was found that high temperature provoked more significant changes in polyamine levels than low temperature. The results suggest that the eti5 mutant showed a better ability to recover after the temperature treatments than wild type partly as a consequence of changes in polyamine content.     

Polyamine metabolism in Ancylostoma ceylanicum and Nippostrongylus brasiliensis

Spermidine was detected as the major polyamine of Ancylostoma ceylanicum as well as Nippostrongylus brasiliensis. Spermine was present in lower amounts whereas the level of putrescine was even less. S-Adenosylmethionine decarboxylase, a rate-limiting enzyme in the biosynthetic pathway of polyamines, was demonstrated at low levels in both parasites. Decarboxylation of lysine and arginine was absent or negligible and that of ornithine questionable, as the enzyme activity was not inhibited by alpha-difluoromethylornithine while RMI 71,645, an irreversible inhibitor of ornithine aminotransferase, strongly inhibited the liberation of CO2 from ornithine. High activity of ornithine aminotransferase was observed in both the parasites and may interfere with the assay for ornithine decarboxylase. Adults of A. ceylanicum were found to rapidly take up spermidine and spermine from incubation medium while uptake of putrescine was very low. These results indicate that hookworms depend on uptake and interconversion rather than de novo synthesis for their polyamine requirement.