Occurrence of uncommon polyamines in cultured tissues of maize

Summary The uncommon polyamines, norspermidine and norspermine, were detected in maizein vitro cultures of three different genotypes. The common polyamines, spermidine and spermine, along with the diamine, putrescine, were also observed. The total amounts of the uncommon polyamines, norspermidine and norspermine, were comparable to the total amounts of the common polyamines, spermidine and spermine, in the maize tissues. The titer for norspermidine was 6- to 15-fold greater than that of its common counterpart (spermidine) in the three genotypes. Norspermidine was the predominant polyamine among all triamines and tetramines detected in cell cultures of two of the three genotypes of maize examined and was predominant along with spermine in the third genotype. Enzyme assays performed with extracts from callus of one of the genotypes suggested a likely mechanism to account for the biosynthesis of the uncommon polyamines in cultured maize cells, through the actions of putrescine aminopropyltransferase, polyamine oxidase, and Schiff-base reductase/decarboxylase enzyme activities. This is the first report of the detection of uncommon polyamines in maize tissues, as well as the first report of these uncommon polyamines in a monocotyledonous plant.     

Crystal structure of Helicobacter pylori spermidine synthase: a Rossmann-like fold with a distinct active site

Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine to putrescine during spermidine biosynthesis. Helicobacter pylori PAPT (HpPAPT) has a low sequence identity with other PAPTs and lacks the signature sequence found in other PAPTs. The crystal structure of HpPAPT, determined by multiwavelength anomalous dispersion, revealed an N-terminal beta-stranded domain and a C-terminal Rossmann-like domain. Structural comparison with other PAPTs showed that HpPAPT has a unique binding pocket between two domains, numerous non-conserved residues, a less acidic electrostatic surface potential, and a large buried space within the structure. HpPAPT lacks the gatekeeping loop that facilitates substrate binding in other PAPTs. PAPTs are essential for bacterial cell viability; thus, HpPAPT may be a potential antimicrobial drug target for H. pylori owing to its characteristic PAPT sequence and distinct conformation.     

Catabolism of polyamines

Summary. Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N¹-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects.Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N⁸-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.     

Polyamines and their biosynthetic enzymes in Ehrlich ascites-carcinoma cells. Modification of tumour polyamine pattern by diamines.

1. Ehrlich ascites-carcinoma cells contained relatively high concentrations of spermidine and spermine, but the putrescine content of the washed cells was less than 10% of that of higher polyamines. 2. Ascites-tumour cells likewise exhibited high activities of L-ornithine decarboxylase (EC 4.1.1.17), S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50), spermidine synthase (EC 2.5.1.16) and spermine synthase. 3. During the first days after the inoculation, the polyamine pattern of the ascites cells was characterized by a high molar ratio of spermidine to spermine, which markedly decreased on aging of the cells. 4. Various diamines injected into mice bearing ascites cells rapidly and powerfully decreased ornithine decarboxylase activity in the carcinoma cells, apparently through a mechanism that was not a direct inhibition of the enzyme in vitro. Cadaverine (1,5-diaminopentane) and 1,6-diaminohexane were the most potent inhibitors of ornithine decarboxylase among the amines tested. 5. Chronic treatment of the mice with diamines resulted in a virtually complete disappearance of ornithine decarboxylase activity, and after 24h a significant decline in spermidine accumulation. 6. Cadaverine appeared to be an especially suitable compound for use as an inhibitor of the synthesis of higher polyamines, at least in Ehrlich ascites cells, since this diamine also acted as a competitive inhibitor for putrescine in the spermidine synthase reaction without being incorporated into the higher polyamines.     

Polyamines as modulators of lipoperoxidation.

The polyamines spermine and spermidine and the diamine putrescine inhibit lipid peroxidation in phospholipid liposome suspensions and rat liver homogenates. Using the chemiluminescence technique the antioxidant activity of polyamines was found to be due to reactions with the free radical intermediates of lipid peroxidation and/or superoxide radicals. Also, the antioxidant action of polyamines correlated with the amount of their amino groups: the antioxidant activity increases from putrescine to spermine.     

Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.

Treatment of rats with spermidine, spermine or sym-norspermidine led to a substantial induction of spermidine/spermine N1-acetyltransferase activity in liver, kidney and lung. The increase in this enzyme, which was determined independently of other acetylases by using a specific antiserum, accounted for all of the increased acetylase activity in extracts from rats treated with these polyamines. Spermine was the most active inducer, and the greatest effect was seen in liver. Liver spermidine/spermine N1-acetyltransferase activity was increased about 300-fold within 6 h of treatment with 0.3 mmol/kg doses of spermine; activity in kidney increased 30-fold and activity in the lung 15-fold under these conditions. The increased spermidine/spermine N1-acetyltransferase activity led to a large increase in the liver putrescine content and a decline in spermidine. These changes are due to the oxidation by polyamine oxidase of the N1-acetylspermidine formed by the acetyltransferase. Our results indicated that spermidine was the preferred substrate in vivo of the acetylase/oxidase pathway for the conversion of the higher polyamines into putrescine. The induction of the spermidine/spermine N1-acetyltransferase by polyamines may provide a mechanism by which excess polyamines can be removed.     

Polyamines as modulators of salt tolerance in rice cultivars

The effect of NaCl on the endogenous levels of diamine, putrescine and polyamines, spermidine and spermine, was studied in the shoot system of salt-tolerant and salt-sensitive lines of rice (Oryza sativa L.) cultivars during three growth stages. Salt stress increased the levels of diamine and polyamine in varying degrees among nine rice cultivars investigated. Salt tolerant AU1, Co43, and CSC1 were effective in maintaining high concentrations of spermidine and spermine, while the content of putrescine was not significantly altered in all the growth stages when plants were exposed to salinity. The salt sensitivity in rice was associated with excessive accumulation of putrescine and with low levels of spermidine and spermine in the shoot system of salt-sensitive cultivars Co36, CSC2, GR3, IR20, TKM4, and TKM9 under saline condition. One of the possible mechanisms of saline resistance was observed to be due to the highly increased polyamines against the low increase in diamines. Alternatively, the salt sensitivity could be due to high increase of diamines and an incapacity to maintain high levels of polyamines.     

Polyamines and environmental challenges: recent development

In this review, we will try to summarize some recent data concerning the changes in polyamine metabolism (biosynthesis, catabolism and regulation) in higher plants subjected to a wide array of environmental stress conditions and to describe and discuss some of the new advances concerning the different proposed mechanisms of polyamine action implicated in plant response to environmental challenges. All the data support the view that putrescine and derived polyamines (spermidine, spermine, long-chained polyamides) may have several functions during environmental challenges. In several systems (except during hypoxia, and chilling tolerance of wheat and rice) an induction of polyamines (spermidine, spermine) not putrescine accumulation, may confer a stress tolerance. In several cases stress tolerance is associated with the production of conjugated and bound polyamines and stimulation of polyamine oxidation. In several environmental challenges (osmotic-stress, salinity, hypoxia, environmental pollutants) recent results indicate that both arginine decarboxylase and ornithine decarboxylase are required for the synthesis of putrescine and polyamines (spermidine and spermine). Under osmotic and salt-stresses a production of cadaverine is observed in plants. A new study demonstrates that under salt-stress putrescine catabolism (via diamine oxidase) can contribute to proline (a compatible osmolyte) accumulation.     

Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 microM increase F(V) as efficiently as 100 microM of spermidine or 1000 microM of putrescine or 1000 microM of Mg(2+). It is also demonstrated that the increase in F(V) derives mainly from the contribution of PSIIalpha centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.     

Effects of Polyamines on Proline Endopeptidase Activity in Rat Brain

The in vitro effects of polyamines on the activity of proline endopeptidase (PEPase) in rat brain cytosol, which contains an endogenous PEPase inhibitor, have been studied. Of the three amines tested (spermine, spermidine, and putrescine), spermine and spermidine markedly enhanced the enzyme activity in brain cytosol. At 6.25 mM spermine or 25 mM spermidine, a 13- or 14-fold enhancement of the enzyme activity was observed. When Mg2+ was used, an approximately fourfold enhancement of the enzyme activity was observed at 50 mM. The enhancement produced by spermine or spermidine was unaffected by Mg2+ up to 50 mM. The activity of purified PEPase was only slightly affected by each polyamine, but it was inhibited 50% by 50 mM Mg2+. On the other hand, 50% inhibition of the enzyme produced by the purified PEPase inhibitor (Mr 7,000: Ki 0.67 mM) was completely restored by addition of 0.7 mM spermine, 3.5 mM spermidine, or 28 mM putrescine. This restoration of inhibition by polyamines was reversed by increasing the inhibitor concentration. These data suggest that polyamines effectively reverse the inhibition of PEPase by its endogenous inhibitor by the reversible formation of a kinetically significant complex. The possible functions of polyamines in the regulation of PEPase in vivo are discussed.     

Polyamines are essential for the synthesis of 2-ricinoleoyl phosphatidic acid in developing seeds of castor

The major fatty acid component of castor (Ricinus communis L.) oil is ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid), and unsaturated hydroxy acid accounts for >85% of the total fatty acids in triacylglycerol (TAG). TAG had a higher ricinoleate content at position 2 than at positions 1 and 3. Although lysophosphatidic acid (LPA) acyltransferase (EC 2.3.1.51), which catalyzes acylation of LPA at position 2, was expected to utilize ricinoleoyl-CoA preferentially over other fatty acyl-CoAs, no activity was found for ricinoleoyl-CoA in vitro at concentrations at which other unsaturated acyl-CoAs were incorporated rapidly. However, activity for ricinoleoyl-CoA appeared with addition of polyamines (putrescine, spermidine, and spermine), while polyamines decreased the rates of incorporation of other acyl-CoAs into position 2. The order of effect of polyamines on LPA acyltransferase activity was spermine > spermidine >> putrescine. At concentrations of spermine and spermidine of >0.1 mM, ricinoleoyl-CoA served as an effective substrate for LPA acyltransferase reaction. The concentrations of spermine and spermidine in the developing seeds were estimated at ∼0.09 and ∼0.63 mM, respectively. These stimulatory effects for incorporation of ricinoleate were specific to polyamines, but basic amino acids were ineffective as cations. In contrast, in microsomes from safflower seeds that do not contain ricinoleic acid, spermine and spermidine stimulated the LPA acyltransferase reaction for all acyl-CoAs tested, including ricinoleoyl-CoA. Although the fatty acid composition of TAG depends on both acyl-CoA composition in the cell and substrate specificity of acyltransferases, castor bean polyamines are crucial for incorporation of ricinoleate into position 2 of LPA. Polyamines are essential for synthesis of 2-ricinoleoyl phosphatidic acid in developing castor seeds.     

Oxidized polyamines and the growth of human vascular endothelial cells. Prevention of cytotoxic effects by selective acetylation.

The responses of human umbilical-vein vascular endothelial cells in culture to the naturally occurring polyamines spermine, spermidine and putrescine, their acetyl derivatives and oxidation products were examined. In the absence of human polyamine oxidase, exposure of cells to polyamines (up to 160 microM) had no adverse effects. In the presence of polyamine oxidase, spermine and spermidine were cytotoxic, but putrescine was not. Acetylation of the aminopropyl group of spermidine or both aminopropyl groups of spermine prevented this cytotoxicity. The amino acids corresponding to the polyamines, representing a further stage of oxidation, were also without effect. The cytotoxic effects were irreversible. Use of bovine serum amine oxidase in place of the human enzyme gave qualitatively similar results.     

Evidence for the occurrence of polyamine oxidase in the dicotyledonous plant Medicago sativa L. (alfalfa)

Polyamine oxidase (EC 1.5.3.3) activity has not been detected previously in cells of dicotyledonous plants, although it has been characterized extensively in monocotyledonous plants. Evidence is presented in this report for the occurrence of polyamine oxidase in dialyzed crude extracts of the dicotyledonous plant, Medicago sativa L. (alfalfa). Three enzyme assays were used to quantitate the formation of the three products of the reaction catalyzed by polyamine oxidase. 1-Pyrroline formation was measured colorimetrically as a yellow quinazolinium complex with o-aminobenzaldehyde. Hydrogen peroxide formation was measured spectrophotometrically with a coupled peroxidase assay system by peroxidative oxidation of guaiacol. [³H]1,3-Diaminopropane formation was measured by using [1,8-³H]spermidine as the substrate and separating the radiolabelled reaction product from the substrate by paper electrophoresis. This latter assay provided evidence that a polyamine oxidase of type [EC 1.5.3.3] catalyzed the cleavage reaction between a secondary nitrogen atom and an adjacent carbon of the butyl moiety of spermidine. Significant polyamine oxidase activity was detected in floral tissues, cortex tissues of the root, young leaves, and young germinated seedlings of alfalfa. The occurrence of polyamine oxidase in alfalfa accounts for the formation of the essential substrate, 1,3-diaminopropane, required for the biosynthesis of the uncommon polyamines, norspermidine and norspermine, which we have recently detected in alfalfa.Abbreviations PAO polyamine oxidase - MOPS [3-(N-morpholino)propanesulfonic acid] - MES [2-(N-morpholino)ethanesulfonic acid] - TES [N-tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid] - BICINE [N,N-bis (2-hydroxyethyl)glycine] - DTC diethyldithiocarbamic acid - R_m the distance of migration of a polyamine relative to putrescine after electrophoresis on paper     

Polyamines and plant disease

The diamine putrescine and the polyamines spermidine and spermine are found in a wide range of organisms from bacteria to plants and animals. They are basic, small molecules implicated in the promotion of plant growth and development by activating the synthesis of nucleic acids. Polyamine metabolism has long been known to be altered in plants responding to abiotic environmental stress and to undergo profound changes in plants interacting with fungal and viral pathogens. Polyamines conjugated to phenolic compounds, hydroxycinnamic acid amides (HCAAs), have been shown to accumulate in incompatible interactions between plants and a variety of pathogens, while changes in the diamine catabolic enzyme diamine oxidase suggest a role for this enzyme in the production of hydrogen peroxide during plant defence responses. More recent work has suggested a role for the free polyamine spermine in the hypersensitive response of barley to powdery mildew and particularly in tobacco to TMV. The prospects for the genetic manipulation of HCAA levels in plants as a means of both defining their role in plant defence and in the generation of disease resistant plants is discussed briefly.     

The biochemistry, genetics, and regulation of polyamine biosynthesis in Saccharomyces cerevisiae

We have studied the enzymes and genes involved in the biosynthesis of putrescine, spermidine, and spermine in Saccharomyces cerevisiae. Mutants have been isolated with defects in the biosynthetic pathway as follows: spe10 mutants, deficient in ornithine decarboxylase, cannot make putrescine, spermidine, or spermine; spe2 mutants, lacking S-adenosylmethionine decarboxylase, cannot make spermidine or spermine; spe3 mutants, lacking putrescine aminopropyltransferase, cannot make spermidine or spermine; and spe4 and spe40 mutants, lacking spermidine aminopropyltransferase, contain no spermine and permit growth of spe10 mutants. Studies with these mutants have shown that in yeast: 1) polyamines are absolutely required for growth; 2) putrescine is formed only by decarboxylation or ornithine; 3) two separate aminopropyltransferases are required for spermidine and spermine synthesis; 4) spermine and spermidine are important in the regulation of ornithine decarboxylase and the amines exert this control by a posttranslational modification of the enzyme; and 5) spermidine or spermine is essential for sporulation of yeast and for the maintenance of the double-stranded RNA killer plasmid. Recent studies in amine-deficient mutants of Escherichia coli have shown an important role of the polyamines in protein synthesis in vivo.     

Acrolein produced from polyamines as one of the uraemic toxins

It is well known that the addition of spermine or spermidine to culture medium containing ruminant serum inhibits cellular proliferation. This effect is caused by the products of oxidation of polyamines that are generated by serum amine oxidase. Among the products, we found that acrolein is a major toxic compound produced from spermine and spermidine by amine oxidase. We then analysed the level of polyamines (putrescine, spermidine and spermine) and amine oxidase activity in plasma of patients with chronic renal failure. It was found that the levels of putrescine and the amine oxidase activity were increased, whereas spermidine and spermine were decreased in plasma of patients with chronic renal failure. The levels of free and protein-conjugated acrolein were also increased in plasma of patients with chronic renal failure. An increase in putrescine, amine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. These results suggest that acrolein is produced during the early stage of nephritis through kidney damage and also during uraemia through accumulation of polyamines in blood due to the decrease in their excretion into urine.     

Reversal by aminoguanidine of the inhibition of proliferation of human fibroblasts by spermidine and spermine.

The inhibitory effect of the polyamines, spermidine and spermine, on the proliferation of human fibroblasts in culture was found to be reversed by the addition of aminoguanidine (AM), a specific and highly effective inhibitor of diamine oxidase (DAO) present in fetal calf serum (FCS). Aminoguanidine itself in concentration as high as 10(-3) M exhibited no effect upon cell proliferation nor did putrescine at similar concentrations. However, at higher concentrations of putrescine, cell proliferation was inhibited and this inhibition was unaffected by the addition of mM concentrations of AM. These studies support earlier hypotheses on the mechanisms of the toxic effects of polyamines on cell proliferation and establish further that the diamine oxidase-catalyzed metabolism of spermine and spermidine is necessary for their toxic effects in cell culture.