Enzymatic Synthesis of a Branched Trisaccharide, 2,4-di-α-Glucosyl-glucose

New procedures for determining putrescine, spermidine and spermine were first established here by the end point assay method using polyamine oxidase from Penicillium chrysogenum or Aspergillus terreus and putrescine oxidase from Micrococcus rubens. Method 1: Spermidine and spermine were first oxidized with polyamine oxidase (step A). To the reaction mixture, putrescine oxidase was added to oxidize putrescine (step B). Putrescine and spermidine in another reaction mixture were oxidized with putrescine oxidase (step C). Method 2 : Putrescine and spermidine were first oxidized with putrescine oxidase (step A). To the reaction mixture, polyamine oxidase was added to oxidize spermine (step B). Spermidine and spermine in another reaction mixture were oxidized with polyamine oxidase (step C). The amounts of putrescine, spermidine and spermine were determined from the absorbance values at each steps A, B and C.     

Seasonal changes of polyamines in habitat adaptation of different ecotypes of reed plants

The leaves of four reed ecotypes (Phragmites communis Trinius) growing in the desert regions of northwest China were investigated for levels of polyamines and activity of arginine decarboxylase (ADC; EC 4.1.1.19) during the growing season of 5 months. The polyamines in the leaves of all reed ecotypes consisted of putrescine, spermidine and spermine. The polyamine levels of the leaves were lower in the swamp reed than in the terrestrial reed ecotypes. Leaf polyamine levels decreased in all ecotypes over the course of the season. Compared to the swamp reed, the terrestrial reed ecotypes maintained higher ADC activity and a predominance of spermine, resulting in a lower ratio of putrescine to spermidine and spermine. It seems that the adaptation of reed plants to drought and saline habitats may be correlated with putrescine synthesis via the ADC pathway, and with a successful conversion of putrescine to spermidine and spermine.     

Effects of external osmolality on polyamine metabolism in HeLa cells

The polyamine content of Escherichia coli is inversely related to the osmolality of the growth medium. The experiments described here demonstrate that a similar phenomenon occurs in mammalian cells. When grown in media of low NaCl concentration, HeLa cells and human fibroblasts were found to contain high levels of putrescine, spermidine, and spermine. The putrescine content of HeLa cells was a function of the osmolality of the medium, as shown by growing cells in media containing mannitol or additional glucose. External osmolality per se had no effect on the contents of spermidine and spermine. For all media, the total cellular polyamine content could be correlated with the activity of ornithine decarboxylase, the first enzyme in polyamine biosynthesis. Different levels of enzyme activity appear to result solely from variations in the rate of enzyme degradation.A sudden increase in NaCl concentration produced rapid loss of ornithine decarboxylase activity and a gradual loss of putrescine and spermidine. A sudden decrease in NaCl concentration led to rapid and substantial increases in ornithine decarboxylase activity and putrescine.     

Comprehensive analysis of polyamine transport and biosynthesis in the dominant human gut bacteria: Potential presence of novel polyamine metabolism and transport genes

Recent studies have reported that polyamines in the colonic lumen might affect animal health and these polyamines are thought to be produced by gut bacteria. In the present study, we measured the concentrations of three polyamines (putrescine, spermidine, and spermine) in cells and culture supernatants of 32 dominant human gut bacterial species in their growing and stationary phases. Combining polyamine concentration analysis in culture supernatant and cells with available genomic information showed that novel polyamine biosynthetic proteins and transporters were present in dominant human gut bacteria. Based on these findings, we suggested strategies for optimizing polyamine concentrations in the human colonic lumen via regulation of genes responsible for polyamine biosynthesis and transport in the dominant human gut bacteria.     

Distribution of polyamines and their related catabolic enzyme in etiolated and light-grown leguminosae seedlings

Diamine-oxidase (DAO; EC 1.4.3.6) activity and di-and polyamine levels were estimated along the epicotyl and root of light-grown and etiolated lentil (Lens culinaris Medicus) and pea (Pisum sativum L.) seedlings. The activity of DAO was higher in etiolated epicotyls than in lightgrown ones. In both species there was a positive correlation between DAO activity and the diamine (putrescine and cadaverine) levels along the whole epicotyl and root. Polyamine (spermine and spermidine) distribution seemed to be associated with the meristematic and elongating zone of the epicotyl and root. The physiological function of DAO is discussed in relation to its possible role in providing hydrogen peroxide to peroxidase-dependent reactions occurring in the cell wall.Abbreviations CAD cadaverine - DA diamine - DAO diamine oxidase - PA polyamine - PUT putrescine - SPD spermidine - SPM spermine     

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.     

Tissue-specific changes in polyamine levels of neural tissue and fat body in adult crickets

The three major polyamines—putrescine, spermidine, and spermine—were studied and changes of their levels were examined in extracts of cerebral ganglia and fat body from adult Acheta domesticus. In nervous tissue, only spermidine and spermine were present and spermine was two- to three-fold more abundant than spermidine. The polyamine levels were high up to day 3, decreased on day 4, and then remained relatively unchanged up to day 10. The spermidine/spermine ratios decreased during the imaginal life. Higher spermidine titres were observed in the neural tissue of egg-laying females compared to virgin females. In the fat body, putrescine was detected together with spermidine and spermine. Spermidine and spermine levels were two-fold higher than putrescine. Fat body of virgin females contained two times more polyamines than male fat body. Low at emergence, spermidine and spermine concentrations peaked on days 2–3 only in females, and egg-laying was characterized by an increase of putrescine and spermidine titres. Starvation did not change polyamine contents, implying homeostatic regulation of the intracellular polyamine metabolism. These data showing tissue specific changes in polyamine levels during the imaginal life of Acheta domesticus point to the physiological importance of polyamines as possible intracellular regulators during adult insect development. © 1993 Wiley-Liss, Inc.     

Catabolism of polyamines in the rat: Polyamines and their non-α-amino acid metabolites

The metabolic fate of stable isotopically labeled polyamines was investigated after their first and second intraperitoneal injection in rats. Using gas chromatographic and mass fragmentographic analyses of acid-hydrolyzed 24-h urines, some aspects of the polyamine metabolism could be elucidated. After the injections with hexadeutero-1,3-diaminopropane, obly labeled 1,3-diaminopropane was recovered from the urine samples. The rat injected with tetradeuteroputrescine excreted labeled putrescine excreted labeled putrescine, γ-amino-n-butyric acid, 2-hydroxyputrescine and spermidine, while the urine samples of the rat after the injections with tetradeuterocadaverine contained labeled cadaverine and δ-aminovaleric acid. The injections of hexadeuterospermidine led to the appearance of labeled spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, γ-amino-n-butyric acid, 1,3-diaminopropane, β-alanine and spermine. After the injections with octadeuterospermine, labeled spermine, N-(3-aminopropyl)-N′-(2-carboxyethyl)-1,4-diaminobutane, N,N′-bis(2-carboxyethyl)-1,4-diaminobutane, spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, 1,3-diaminopropane, β-alanine, 2-hydroxyputrescine and possibly γ-amino-n-butyric acid were recovered. Clear differences between the metabolism after the first and second injection were noted for putrescine, spermidine and spermine, which is suggestive for enzyme induction and/or the existence of salvage pathways.     

Polyamine dependence of Chinese hamster ovary cells in serum-free culture is due to deficient arginase activity

We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine- and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed.     

Stimulating effect of spermine on bulblet formation in bulb-scale segments of Lilium longiflorum

When bulb-scale segments of Lilium longiflorum were cultured on a medium containing auxin and cytokinin, the proportion of the expiants with newly-formed bulblets was significantly increased by the application of different polyamines. The most effective polyamine was spermine, where more than 90% of segments formed an average of 5 bulblets as compared to controls where less than 50% explants formed an average of 1.5 bulblets. Application of arginine one of the precursors putrescine biosynthesis, slightly promoted bulblet formation. The putrescine-stimulated bulblet formation was strongly inhibited by simultaneous addition of an inhibitor of the spermidine synthase, cyclohexylamine. The spermidine-promoted bulblet formation, however, could not be suppressed by this inhibitor. The promotive effect of spermidine on bulblet formation was reversed by an inhibitor of the spermine synthase, N-(3-aminopropyl)cyclohexylamine, but application of this inhibitor with spermine did not show any apparent effect on the bulblet formation. Endogenous level of spermine increased in common during bulblet formation that were stimulated by exogenous polyamines. Thus, spermine seemed to be the main stimulating chemical on bulblet formation in lily bulb-scale segments.Abbreviations APCHA N-(3-aminopropyl)cyclohexylamine - Arg arginine - BA benzyladenine - CHA cyclohexylamine - MS Murashige and Skoog's - NAA naphthaleneacetic acid - Orn ornithine - Put putrescine - Spd spermidine - Spm spermine     

Polyamines and Morphogenesis - Effects of Methylglyoxal-bis(guanylhydrazone)

The effects of methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis were studied on tuberization and cellular polyamine content using in vitro Solanum tuberosum (cv Binjte) plants. When MGBG was added to the culture medium, it produced a partial inhibition of the growth of stems and leaves; it totally blocked rhizogenesis and strongly stimulated tuber formation. Morphogenetic effects of MGBG were correlated to a 40 % decrease in free putrescine, spermidine, spermine content of the leaves and to a 28 % decrease in spermidine titer of the stems. In the tubers, this inhibitor did not change the free polyamine titer but increased by up to 85 % the titer of conjugated putrescine, spermidine, spermine. When the plants were grown in the dark, MGBG produced, like benzyladenine, a stimulation of the rate of tuberization and enhanced the content of conjugated polyamines in the tuber. These results support the hypothesis that polyamines play an important role in the morphogenesis of potato plants. The role of polyamine conjugation in tuber development is discussed.     

Polyamine depletion delays apoptosis of rat intestinal epithelial cells

The polyamines spermidine, spermine, and their precursorputrescine are essential for cell growth and the regulation of the cellcycle. Recent studies suggest that excessive accumulation of polyaminesfavors either malignant transformation or apoptosis, depending on thecell type and the stimulus. This study examines the involvement ofpolyamines in the induction of apoptosis by the DNA topoisomerase Iinhibitor, camptothecin. In IEC-6 cells, camptothecin induced apoptosiswithin 6 h, accompanied by detachment of cells. Detached cells showedDNA laddering and caspase 3 induction, characteristic features ofapoptosis. Depletion of putrescine, spermidine, and spermine byDL--difluoromethylornithine (DFMO), a specific inhibitorof ornithine decarboxylase (ODC) that is the first rate-limiting enzymefor polyamine biosynthesis, decreased the apoptotic index. Delayedapoptosis was accompanied by a decrease in caspase 3 activity inpolyamine-depleted cells. Addition of putrescine restored the inductionof apoptosis as indicated by an increase in the number of detachedcells and caspase 3 activity. Polyamine depletion did not change thelevel of caspase 3 protein. Inhibition of S-adenosylmethioninedecarboxylase by a specific inhibitor [diethylglyoxalbis-(guanylhydrazone); DEGBG] led to depletion of spermidine andspermine with a significant accumulation of putrescine and induction ofODC. The DEGBG-treated cells showed an increase in apoptosis,suggesting the importance of putrescine in the apoptotic process.Addition of putrescine to DFMO-treated cell extracts did not increasecaspase 3 activity. The above results indicate that polyamine depletiondelays the onset of apoptosis in IEC-6 cells and confers protectionagainst DNA damaging agents, suggesting that polyamines might beinvolved in the caspase activating signal cascade.

     

Senescence of Rice Leaves XXX Levels of Endogenous Polyamines and Dark-Induced Senescence of Rice Leaves

The role of endogenous polyamines in the control of dark-inducedsenescence of detached rice leaves was investigated by quantitatinglevels of various polyamines by HPLC. Putrescine, spermidineand spermine were all present throughout senescence. Neithercadaverine nor 1,3-diaminopropane was detected. During dark-inducedsenescence, there was a marked decrease in levels of putrescineand an increase in those of spermidine and spermine. The rateof production of ethylene increased markedly upon excision ofleaves. -Difluoromethylarginine (DFMA) and -difluoromethylornithine(DFMO) caused a reduction in levels of putrescine, yet had noeffect on levels of spermidine and spermine. Neither DFMA norDFMO had any effect on senescence or on the production of ethylene.Treatment with dicyclohexylamine (DCH) and methylglyoxal bis-(guanylhydrazone)(MGBG) reduced levels of spermine and increased those of putrescinein detached leaves. After treatment with DCH or MGBG, both senescenceand the production of ethylene were significantly promoted.The current results suggest that endogenous polyamines may notplay a significant role in the control of dark-induced senescenceof rice leaves. This conclusion is supported by the furtherobservations that (a) benzyladenine, which is known to retardsenescence, decreased levels of putrescine but had no effecton those of spermidine and spermine; and (b) ABA, which promotedsenescence, increased levels of putrescine and had no effecton those of spermidine and spermine. (Received March 30, 1991; Accepted June 27, 1991)     

Oxidation of Polyamines by Fungal Enzymes

Polyamine oxidase was found in mycelia of fungi belonging to the genera of Aspergillus, Mucor, Penicillium, Rhizopus, Cylindrocarpon, Fusarium and Gibberella when they were grown in medium containing spermine or spermidine as the sole source of nitrogen. The maximal formation of the enzymes of Penicillium chrysogenum and Aspergillus terreus was observed in early stationary phase of growth, and thereafter, the enzymes disappeared with consumption of substrate. The oxidation products of spermine and spermidine by the two enzymes were identified as putrescine, 3-aminopropionaldehyde and H₂O₂. Therefore, the enzymes were characterized as a type of polyamine oxidase of rat liver.     

Polyamine metabolism during sclerotial development of Sclerotinia sclerotiorum

A study on polyamine metabolism and the consequences of polyamine biosynthesis inhibition on the development of Sclerotinia sclerotiorum sclerotia was conducted. Concentrations of the triamine spermidine and the tetramine spermine, as well as ornithine decarboxylase and S-adenosyl-methionine decarboxylase activities, decreased during sclerotia maturation. In turn, the concentration of the diamine putrescine was reduced at early stages of sclerotial development but it increased later on. This increment was not related to de novo biosynthesis, as demonstrated by the continuous decrease in ornithine decarboxylase activity. Alternatively, it could be explained by the release of putrescine from the conjugated polyamine pool. α-Difluoro-methylornithine and cyclohexylamine, which inhibit putrescine and spermidine biosynthesis, respectively, decreased mycelial growth, but did not reduce the number of sclerotia produced in vitro even though they disrupted polyamine metabolism during sclerotial development. It can be concluded that sclerotial development is less dependent on polyamine biosynthesis than mycelial growth, and that the increase of free putrescine is a typical feature of sclerotial development. The relationship between polyamine metabolism and sclerotial development, as well as the potential of polyamine biosynthesis inhibition as a strategy for the control of plant diseases caused by sclerotial fungi are discussed.     

Polyamine Metabolism in Acanthamoeba: Polyamine Content and Synthesis of Ornithine,Putrescine, and Diaminopropane1

Five polyamines which could be separated by high performance liquid chromatography were found in Acanthamoeba castellanii (strain Neff). These included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. Only diaminopropane and norspermidine had been found previously. Spermine was present in cultures grown in broth, but not in defined medium. Radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ornithine, ornithine was synthesized from arginine or citrulline, and diaminopropane was synthesized from spermidine. The presence of ornithine decarboxylase (EC 4.1.1.17), arginase (EC 3.5.3.1), and urease (EC 3.5.1.5) and the absence of arginine decarboxylase (EC 4.1.1.19) were established. A scheme for polyamine biosynthesis in A. castellanii is proposed.     

Spermine facilitates recovery from drought but does not confer drought tolerance in transgenic rice plants expressing <Emphasis Type="Italic">Datura stramonium</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine decarboxylase

Polyamines are known to play important roles in plant stress tolerance but it has been difficult to determine precise functions for each type of polyamine and their interrelationships. To dissect the roles of putrescine from the higher polyamines spermidine and spermine, we generated transgenic rice plants constitutively expressing a heterologous S-adenosylmethionine decarboxylase (SAMDC) gene from Datura stramonium so that spermidine and spermine levels could be investigated while maintaining a constant putrescine pool. Whereas transgenic plants expressing arginine decarboxylase (ADC) produced higher levels of putrescine, spermidine and spermine, and were protected from drought stress, transgenic plants expressing SAMDC produced normal levels of putrescine and showed drought symptoms typical of wild type plants under stress, but the transgenic plants showed a much more robust recovery on return to normal conditions (90% full recovery compared to 25% partial recovery for wild type plants). At the molecular level, both wild type and transgenic plants showed transient reductions in the levels of endogenous ADC1 and SAMDC mRNA, but only wild type plants showed a spike in putrescine levels under stress. In transgenic plants, there was no spike in putrescine but a smooth increase in spermine levels at the expense of spermidine. These results confirm and extend the threshold model for polyamine activity in drought stress, and attribute individual roles to putrescine, spermidine and spermine.     

Polyamine changes during the development of zygotic embryos in Prunus avium

A study of the polyamine profile was carried out during zygotic embryo development in Prunus avium. Zygotic embryos were collected from 2 donor trees and sorted into 3 size classes: C1 [2.5 to 3.5 mm]; C2 [3.6 to 4.5 mm] and C3 [5.5 to 7 mm]. Evolution of the various polyamines was similar for the two donor trees. Changes in the relative amount of the various free polyamines were observed during zygotic embryo development. Agmatine and spermine levels increased from C1 to C3. Spermidine, the predominant polyamine, showed a two-fold decrease in C3 compared with C1 and C2; the evolution of putrescine was opposed, showing an increase in the last developmental stage. The putrescine/spermidine ratio could be a marker for these 3 developmental stages with a higher ratio in C3 compared with C1 and C2. Polyamine changes in cotyledons from class C1 were investigated during in vitro culture. A 10-day induction on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin caused a strong decline in free spermidine levels and a dramatic increase in free putrescine. The formation of conjugated putrescine occurred simultaneously, and twenty days after removal of growth regulators, the various polyamine contents were still at the same level.Abbreviations Agm agmatine - Dap diaminopropane - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine - Spm spermine     

Effect of organic and inorganic nitrogen sources on endogenous polyamines and growth of ectomycorrhizal fungi in pure culture

 The abilities of three ectomycorrhizal fungi, Paxillus involutus, Suillus variegatus and Lactarius rufus, to utilize organic and inorganic nitrogen sources were determined by measuring the growth and endogenous free polyamines (putrescine, spermidine and spermine) of pure culture mycelium. Differences were found in the utilization of the nitrogen sources and in the polyamine concentrations between the fungal species and between isolates of L. rufus. All the fungi grew well on ammonium and on several amino acids. Endogenous polyamine levels varied with the nitrogen source. Spermidine was commonly the most abundant polyamine; however, more putrescine than spermidine was found in P. involutus growing on inorganic nitrogen or arginine. Low amounts of spermine were found in S. variegatus and some samples of L. rufus. None or only a trace of spermine was found in P. involutus mycelium. In all fungi, putrescine concentrations were higher with ammonium than with the nitrate treatment. The total nitrogen content of peat did not determine the ability of L. rufus strains isolated from peatland forest sites to utilize organic nitrogen. Accepted: 27 November 1998