Polyamine metabolism in <Emphasis Type="Italic">Leishmania</Emphasis>: from arginine to trypanothione

Polyamines (PAs) are essential metabolites in eukaryotes, participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. The PAs are synthesized by a metabolic process which involves arginase (ARG), which catalyzes the enzymatic hydrolysis of l-arginine (l-Arg) to l-ornithine and urea, and ornithine decarboxylase (ODC), which catalyzes the enzymatic decarboxylation of l-ornithine in putrescine. The S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the irreversible decarboxylation of S-adenosylmethionine (AdoMet), generating the decarboxylated S-adenosylmethionine (dAdoMet), which is a substrate, together with putrescine, for spermidine synthase (SpdS). Leishmania parasites and all the other members of the trypanosomatid family depend on spermidine for growth and survival. They can synthesize PAs and polyamine precursors, and also scavenge them from the microenvironment, using specific transporters. In addition, Trypanosomatids have a unique thiol-based metabolism, in which trypanothione (N1-N8-bis(glutathionyl)spermidine, T(SH)₂) and trypanothione reductase (TR) replace many of the antioxidant and metabolic functions of the glutathione/glutathione reductase (GR) and thioredoxin/thioredoxin reductase (TrxR) systems present in the host. Trypanothione synthetase (TryS) and TR are necessary for the protozoa survival. Consequently, enzymes involved in spermidine synthesis and its utilization, i.e. ARG, ODC, AdoMetDC, SpdS and, in particular, TryS and TR, are promising targets for drug development.     

Characterization of transgenic mice with overexpression of spermidine synthase

A composite cytomegalovirus-immediate early gene enhancer/chicken β-actin promoter (CAG) was utilized to generate transgenic mice that overexpress human spermidine synthase (SpdS) to determine the impact of elevated spermidine synthase activity on murine development and physiology. CAG-SpdS mice were viable and fertile and tissue SpdS activity was increased up to ninefold. This increased SpdS activity did not result in a dramatic elevation of spermidine or spermine levels but did lead to a 1.5- to 2-fold reduction in tissue spermine:spermidine ratio in heart, muscle and liver tissues with the highest levels of SpdS activity. This new mouse model enabled simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic animals. The combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair viability or lead to overt developmental abnormalities but instead normalized the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. The CAG-SpdS mice were bred to MHC-AdoMetDC mice with a >100-fold increase in cardiac S-adenosylmethionine decarboxylase (AdoMetDC) activity to determine if elevated dcAdoMet would facilitate greater spermidine accumulation in mice with SpdS overexpression. CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are not rate limiting in vivo for polyamine biosynthesis and are unlikely to exert significant regulatory effects on cellular polyamine content and function.     

Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, ofS-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.     

Biosynthesis of spermidine, a direct precursor of pyrrolizidine alkaloids in root cultures of Senecio vulgaris L.

 The polyamine spermidine is an essential biosynthetic precursor of pyrrolizidine alkaloids. It provides its aminobutyl group which is transferred to putrescine yielding homospermidine, the specific building block of the necine base moiety of pyrrolizidine alkaloids. The enzymatic formation of spermidine was studied in relation to the unique role of this polyamine as an alkaloid precursor. S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and spermidine synthase (SPDS, EC 2.5.1.16) from root cultures of Senecio vulgaris were partially purified and characterized. The SAMDC-catalyzed reaction showed a pH optimum of 7.5, that of SPDS an optimum of 7.7. The K _m value of SAMDC for its substrate S-adenosylmethionine (SAM) was 15 μM, while the apparent K _m values of SPDS for its substrates decarboxylated SAM (dSAM) and putrescine were 4 μM and 21 μM, respectively. The relative molecular masses of the two enzymes, determined by gel filtration, were 29 000 (SAMDC) and 37 000 (SPDS). Studies with various potential inhibitors revealed, for most inhibitors, profiles that were similar to those established with the respective enzymes from other plant sources. However, putrescine which is not known to be an inhibitor of plant SAMDC, strongly inhibited the enzyme from S. vulgaris roots. Spermidine synthase was sensitive to inhibition by its product spermidine. In the presence of the stationary tissue concentrations of the two polyamines (ca. 0.1 mM each) the activities of SAMDC and SPDS would be inhibited by >80%. The results are discussed in relation to the role of spermidine in primary and secondary metabolism of alkaloid-producing S. vulgaris root cultures. Received: 15 September 1999 / Accepted 10 December 1999     

Putrescine N-methyltransferase – The start for alkaloids

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development.     

Putrescine <Emphasis Type="Italic">N</Emphasis>-methyltransferases—a structure–function analysis

Putrescine N-methyltransferase (PMT) is a key enzyme of plant secondary metabolism at the start of the specific biosynthesis of nicotine, of tropane alkaloids, and of calystegines that are glycosidase inhibitors with nortropane structure. PMT is assumed to have developed from spermidine synthases (SPDS) participating in ubiquitous polyamine metabolism. In this study decisive differences between both enzyme families are elucidated. PMT sequences were known from four Solanaceae genera only, therefore additional eight PMT cDNA sequences were cloned from five Solanaceae and a Convolvulaceae. The encoded polypeptides displayed between 76% and 97% identity and typical amino acids different from plant spermidine synthase protein sequences. Heterologous expression of all enzymes proved catalytic activity exclusively as PMT and K _cat values between 0.16 s⁻¹ and 0.39 s⁻¹. The active site of PMT was initially inferred from a protein structure of spermidine synthase obtained by protein crystallisation. Those amino acids of the active site that were continuously different between PMTs and SPDS were mutated in one of the PMT sequences with the idea of changing PMT activity into spermidine synthase. Mutagenesis of active site residues unexpectedly resulted in a complete loss of catalytic activity. A protein model of PMT was based on the crystal structure of SPDS and suggests that overall protein folds are comparable. The respective cosubstrates S-adenosylmethionine and decarboxylated S-adenosylmethionine, however, appear to bind differentially to the active sites of both enzymes, and the substrate putrescine adopts a different position.     

Effects of inhibitors of spermidine and spermine synthesis on polyamine concentrations and growth of transformed mouse fibroblasts

1. A number of compounds known to inhibit polyamine biosynthesis at various steps in the biosynthetic pathway were tested for their ability to inhibit growth and decrease polyamine concentrations in virally transformed mouse fibroblasts (SV-3T3 cells). 2. Virtually complete inhibition of growth was produced by the inhibitors of ornithine decarboxylase α-methylornithine and α-difluoromethylornithine and by the inhibitors of S-adenosylmethionine decarboxylase 1,1′-[(methylethanediylidene)dinitrilo]diguanidine and 1,1′-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine). The former inhibitors decreased putrescine and spermidine contents in the cells to very low values, whereas the latter substantially increased putrescine but decreased spermidine concentrations. The inhibitory effects of all of these inhibitors on cell growth could be prevented by the addition of spermidine, suggesting that spermidine depletion is the underlying cause of their inhibition of growth. 3. α-Difluoromethylornithine, which is an irreversible inhibitor of ornithine decarboxylase, was a more potent inhibitor of growth and polyamine production (depleting spermidine almost completely and spermine significantly) than α-methylornithine, which is a competitive inhibitor. This was not the case with the inhibitors of S-adenosylmethionine decarboxylase where 1,1′-[(methylethanediylidene)dinitrilo]diguanidine, a reversible inhibitor, was more active than 1,1′-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine), an irreversible inhibitor. It is suggested that this effect may be due to the lesser uptake and/or greater chemical reactivity of the latter compound. 4. Various nucleoside derivatives of S-adenosylhomocysteine that inhibited spermidine synthase in vitro did not have significant inhibitory action against polyamine accumulation in the cell. These compounds, which included S-adenosylhomocysteine sulphone, decarboxylated S-adenosylhomocysteine sulphone, decarboxylated S-adenosylhomocysteine sulphoxide and S-adenosyl-4-thio-butyric acid sulphone did not inhibit cell growth or polyamine content until cytotoxic concentrations were added. 5. 5′-Methylthioadenosine, 5′-isobutylthioadenosine and 5′-methylthiotubercidin, which inhibit aminopropyltransferase activity in vitro, all inhibited cell growth and decreased spermidine content. Although these compounds were most active against spermine synthase in vitro, they acted in the cell primarily to decrease spermidine content. Cell growth could not be restored to normal values by addition of spermidine, suggesting that these nucleosides have another inhibitory action towards cellular proliferation. 6. 5′-Methylthioadenosine and 5′-isobutylthioadenosine are degraded by a phosphorylase present in SV3T3 cells, yielding 5-methylthioribose-1-phosphate and 5-isobutylthioribose-1-phosphate respectively, and adenine. This degradation appears to decrease the inhibitory action towards cell growth, suggesting that the nucleosides themselves are exerting the inhibitory action. 5′-Methylthiotubercidin, which is not a substrate for the phosphorylase and is a competitive inhibitor of it, was the most active of these nucleosides in inhibiting cell growth and spermidine content. 5′-Methylthiotubercidin and α-difluoromethylornithine had additive effects on retarding cell growth, but not on cellular spermine accumulation, also suggesting that the primary growth-inhibiting action of the nucleoside was not on polyamine production. 7. These results support the concept that 5′-methylthioadenosine phosphorylase plays an important role in permitting cell growth to continue by preventing the build-up of inhibitory intracellular concentrations of 5′-methylthioadenosine.     

Polyamine metabolism in barley reacting hypersensitively to the powdery mildew fungus Blumeria graminis f. sp. hordei

Polyamine levels and activities of enzymes of polyamine biosynthesis and catabolism were examined in the barley cultivar Delibes (Ml1al + Ml(Ab)) reacting hypersensitively to the powdery mildew fungus, Blumeria graminis f. sp. hordei (race CC220). Levels of free putrescine and spermine and of conjugated forms of putrescine, spermidine and spermine were greatly increased 1–4 d following inoculation of barley with the powdery mildew. These changes in polyamine levels were accompanied by elevated activities of the polyamine biosynthetic enzymes ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and S‐adenosylmethionine decarboxylase (AdoMetDC) and the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO). Activities of two enzymes involved in conjugating polyamines to hydroxycinnamic acids, putrescine hydroxycinnamoyl transferase (PHT) and tyramine feruloyl‐CoA transferase (TFT) were also examined and were found to increase significantly 1–4 d after inoculation. The possibility that the increased levels of free spermine, increased polyamine conjugates, and increased DAO and PAO activities are involved in development of the hypersensitive response of Delibes to powdery mildew infection is discussed.     

Effects of polyamine biosynthesis inhibitors on the progesterone-initiated increase in intracellular free Ca2+ and acrosome reactions in human sperm

This laboratory has previously reported that progesterone can initiate a rapid transient increase in the concentration of intracellular free Ca²⁺([Ca²⁺]_i) and an increase in a Ca²⁺-requiring exocytotic event, the acrosome reaction (AR) in human sperm. Rapid increases in Ca²⁺ fluxes of some mammalian cells caused by another steroid, testosterone, require polyamine biosynthesis. Herein, we tested two polyamine biosynthesis suicide inhibitors for their effects on the progesterone-initiated increase in [Ca²⁺]_i and AR in capacitated human sperm in vitro: DL-α-(difluoromethyl)ornithine hydrochloride (DFMO), an inhibitor of putrescine synthesis by ornithine decarboxylase and (5′-{[(Z))-4-amino-2-butenyl]methylamino}-5′-deoxyadenosine (MDL 73811), an inhibitor of S-adenosylmethionine decarboxylase (required for spermidine and spermine synthesis). Sperm were capacitated in vitro and preincubated 10 min with 4.9 mM DFMO or 9.8 μM MDL 73811 with or without various polyamines (245 μM). Progesterone (3.09 μM final concentration) or progesterone solvent (ethanol, 0.1% final concentration) was then added, sperm fixed 1 min after additions and AR assayed by indirect immunofluorescence or with fluorescein-labeled Con A lectin. DFMO strongly inhibited the AR but putrescine (product of ornithine decarboxylase and precursor of spermidine and spermine) reversed that inhibition. Preincubation for 25 min with DMFO + spermidine also reversed DFMO inhibition. MDL 73811 inhibited the progesterone-initiated AR, and a 10 min preincubation with spermidine, but not putrescine or spermine, reversed that inhibition. Preincubations with putrescine alone or with spermidine alone followed by addition of the progesterone solvent did not initiate the AR, and such preincubations followed by progesterone addition did not increase the AR more than progesterone alone. MDL 73811 and DFMO partially inhibited the rapid progesterone-initiated increase in [Ca²⁺]_i (assayed with fura-2), and those inhibitions were partially reversed by putrescine and spermidine, respectively. Putrescine or spermidine alone did not increase [Ca²⁺]_i nor did preincubation with either polyamine followed by progesterone addition increase [Ca²⁺]_i more than progesterone alone. Neither inhibitor was able to inhibit the AR initiated by the calcium ionophore, ionomycin. Our results suggest that human sperm polyamine biosynthesis is necessary for the progesterone-initiated rapid increase in [Ca²⁺]_i and subsequent membrane events of the AR. © 1993 Wiley-Liss, Inc.     

Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes

Spermidine synthase (EC 2.5.1.16) was purified to homogeneity for the cytosol of soybean (Glycine max) axes using ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, ω-aminooctyl-Sepharose and ATPA-Sepharose. The molecular mass of the enzyme estimated by gel filtration and SDS–PAGE is 74 kDa. Cadaverin and 1,6-diaminohexane could not replace putrescine as the aminopropyl acceptor. Kinetic behaviors of the substrate are consistent with a ping pong mechanism. The kinetic mechanism is further supported by direct evidence confirming the presence of an aminopropylated enzyme and identification of product, 5′-deoxy-5′-methylthioadenosine, prior to adding putrescine. The K_m values for decarboxylated S-adenosylmethionine and putrescine are 0.43 μM and 32.45 μM, respectively. Optimum pH and temperature for the enzyme reaction are 8.5 and 37°C, respectively. The enzyme activity is inhibited by N-ethylmaleimide and DTNB, but stimulated by Co²⁺, Cu²⁺ and Ca²⁺ significantly, suggesting that these metal ions could be the cellular regulators in polyamine biosynthesis.     

Molecular characterization and homology modeling of spermidine synthase from <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7942

Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37?°C. The enzyme had higher affinity for dcSAM (K _m, 20 µM) than for putrescine (K _m, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine.     

Polyamines and ethylene in relation to adventitious root formation in Prunus avium shoot cultures

An attempt was made to identify some of the hormonal factors that control adventitious root formation in our Prunus avium micropropagation system in order to improve rooting in difficult-to-root genotypes. Changes in endogenous contents of free polyamines were determined at intervals during auxin-induced rooting of shoot cultures. Accumulation of putrescine and spermidine peaked between days 9 and 11. Spermine was only present in traces, Exogenously supplied putrescine or spermine (50-500 μM), in the presence of optimal or suboptimal levels of indolebutyric acid (IBA), had no effect on rooting percentage or root density, except for spermine at 500 μM. At this external concentration spermine caused a substantial accumulation in both free spermine and putrescine. The use of several inhibitors of polyamine biosynthesis, namely α-difluoromethylornithine (DFMO), α-difluoromethylarginine (DFMA), dicyclohexylammonium sulphate (DCHA) and methylglyoxal-bis-guanyl-hydrazone (MGBG) alone or in combination in the 0.1 to 5 μM range, resulted in an inhibition of rooting that was partially reversed by the addition of the corresponding polyamine. Cellular polyamine levels were significantly reduced by DFMO and DFMA but not by DCHA and MGBG, Labeled putrescine incorporation into spermidine increased somewhat in the presence of the ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG). A system based on [3,4-¹⁴C]methionine incorporation was used to measure ethylene synthesis by the in vitro cultured shoots. Label incorporation was drastically reduced by 10 μM AVG and increased 3.5-fold in the presence of 50 μM IBA with respect to controls (no IBA). Labeled methionine incorporation into spermidine increased to some extent when ethylene synthesis was inhibited by AVG. Adding the ethylene precursor 1-aminocyclopropane-l-carboxylic acid (ACC) to the rooting medium significantly inhibited rooting percentage; AVG caused the formation of a greater number of roots per shoot but delayed their growth. Supplying the shoots with both compounds resulted in an intermediate rooting response, in which both rooting percentage and root density were affected. These results indicate that polyamines may play a significant role at least in some stages of root formation. The polyamine and ethylene biosynthetic pathways seem to be competitive but under our conditions, the enhancement of one pathway when the other was inhibited, was not dramatic. Although IBA promoted ethylene synthesis, AVG, which drastically reduced it, also promoted root formation. Thus, the auxin effect on root induction cannot be directly related to its ability to enhance ethylene synthesis.     

Propylamine transferases in chinese cabbage leaves

We have found spermidine synthase and spermine synthase activities in extracts of leaves of Chinese cabbage (Brassica pekinensis var. Pak Choy) and have developed an assay of the former in crude extracts. The method is based on the transfer of the propylamine moiety of decarboxylated S-adenosylmethionine to labeled putrescine, followed by ion-exchange separation of the labeled amine substrate and product, which are then converted to the 5-dimethylamino-1-napthalene sulfonyl (dansyl) derivatives and further purified and identified by thin layer chromatography. The specific radioactivity of putrescine present in the reaction mixture is determined, as is the radioactivity present in dansyl spermidine. The enzyme is also present in extracts of spinach leaves.

Spermidine synthase has been purified about 160-fold from Chinese cabbage leaves. After partial purification, a rapid coupled enzymic assay has been used to study various properties of the enzyme. The plant enzyme shows maximum activity at pH 8.8 in glycine-NaOH buffer and has a molecular weight of 81,000. The K_m values for decarboxylated S-adenosylmethionine and putrescine are 6.7 and 32 micromolar, respectively. The enzyme activity is inhibited strongly by dicyclohexylamine, cyclohexylamine, and S-adenosyl-3-thio-1, 8-diaminoctane. Of these, dicyclohexylamine is the most potent inhibitor with an I₅₀ at 0.24 micromolar.

     

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.     

A specific role for spermidine in the initiation phase of somatic embryogenesis in Panax ginseng CA Meyer

Somatic embryogenesis of Panax ginseng CA Meyer was initiated from suspension aggregates of an embryogenic callus, in a liquid medium consisting of half strength Murashige and Skoog (1962) supplemented with the synthetic auxin benzoselenienyl-3 acetic acid. The addition of spermidine to this initiation medium significantly increased the production of somatic embryos. In this case, the total polyamine content of the embryogenic mass was higher than that of cultures without spermidine. At day 6 of the culture, a transient accumulation of free polyamines, mainly spermidine, was observed. After this peak, free and conjugated polyamines levels did not show significant variation nor did the polyamine oxidase activity. The results clearly demonstrated that spermidine supplied to the medium was oxidised by polyamine oxidase and partially metabolised into putrescine. The role of spermidine and its interaction with auxin in the initiation of the embryogenic process in Panax ginseng are discussed in relation to embryogenic potential.     

Structure and mechanism of spermidine synthases

Aminopropyltransferases transfer aminopropyl groups from decarboxylated S-adenosylmethionine to amine acceptors, forming polyamines. Structural and biochemical studies have been carried out with the human spermidine synthase, which is highly specific for putrescine as the amine acceptor, and the Thermotoga maritima spermidine synthase, which prefers putrescine but is more tolerant of other substrates. Comparison of the structures of the human spermidine synthase with both substrates and products with the known structure of T. maritima spermidine synthase complexed to a multisubstrate analogue inhibitor and analysis of the properties of site-directed mutants provide a general mechanistic hypothesis for the aminopropyl transfer reaction. The studies also provide a structural basis for the specificity of the spermidine synthase subclass of the aminopropyltransferase family.     

Effect of inhibition of polyamine synthesis on the content of decarboxylated S-adenosylmethionine

1. The content of decarboxylated S-adenosylmethionine (AdoMet) in transformed mouse fibroblasts (SV-3T3 cells) was increased 500-fold to about 0.4fmol/cell when ornithine decarboxylase was inhibited by α-difluoromethylornithine. This increase was due to the absence of putrescine and spermidine, which serve as substrates for aminopropyltransferases with decarboxylated AdoMet as an aminopropyl donor, and to the enhanced activity of AdoMet decarboxylase brought about by depletion of spermidine. The increase in decarboxylated AdoMet content was abolished by addition of putrescine, but not by 1,3-diaminopropane. 2. 5′-Methylthiotubercidin also increased decarboxylated AdoMet content, presumably by direct inhibition of aminopropyl-transferase activities, but the increase in its content and the decline in spermidine content were much less than those produced by α-difluoromethylornithine. 3. Decarboxylated AdoMet content of regenerating rat liver was measured in rats treated with inhibitors of ornithine decarboxylase. The content was increased by 60% 32h after partial hepatectomy in control rats, by 90% when α-difluoromethylornithine was given to the partially hepatectomized rats, and by 330% when 1,3-diaminopropane was used to inhibit putrescine and spermidine synthesis. After 48h of exposure to 1,3-diaminopropane, which completely prevented the increase in spermidine after partial hepatectomy, there was a 5-fold rise in hepatic decarboxylated AdoMet concentration. These increases were prevented by treatment with putrescine or with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase. 4. These results show that changes in AdoMet metabolism result from the administration of specific inhibitors of polyamine synthesis. The possible consequences of the accumulation of decarboxylated AdoMet, which could, for example, interfere with normal cellular methylation or lead to depletion of cellular adenine nucleotides, should be considered in the interpretation of results obtained with such inhibitors.