Increase In S-adenosylmethionine Decarboxylase Activity Research Articles

Exogenous spermidine regulates the anaerobic enzyme system through hormone concentrations and related-gene expression in Phyllostachys praecox roots under flooding stress

PurposeAcclimation to hypoxia and anoxia is important in various ecological systems, especially flooded soil. Phyllostachys pracecox is sensitive to flooding, and therefore, it is important to explore ways of alleviating hypoxia stress in the roots. In this study, we investigated the regulatory effect of spermidine (Spd) on flooded P. praecox seedlings. MethodsA batch experiment was carried out in roots treated with Spd under flooding for eight days. The following factors were subsequently measured: growth, survival rate, root respiratory activity, soluble protein and anaerobic respiration enzyme contents (pyruvate decarboxylase, PDC; alcohol dehydrogenase, ADH; lactate dehydrogenase, LDH; alanine aminotransferase, AlaAT), S-adenosylmethionine decarboxylase (SAMDC), nitrate reductase (NR), ACC oxidase (ACO) and ACC synthetase (ACS) activities, free Spd, spermine (Spm) and the diamine precursor putrescine (Put) content, indole-3-acetic acid (IAA) and abscisic acid (ABA) content, ethylene emissions and expression of hormone-related genes. ResultsApplication of Spd promoted root growth (root length, volume, surface and dry weight) and root respiratory inhibition, improved the soluble protein content, and reduced the O2·- production rate, H2O2 and MDA content to alleviate the damage of roots under flooding. A significant increase in SAMDC activity, and ABA and IAA contents were also observed, along with a reduction in ethylene emissions, NR, ACO and ACS activities (p < 0.05). Exogenous Spd increased the free Spd and Spm contents in the P. praecox roots, but decreased the free Put content. Taken together, these findings suggest that hypoxia stress was alleviated. Moreover, exogenous Spd up-regulated the expression of auxin-related genes ARF1, AUX1, AUX2, AUX3 and AUX4, and down-regulated the expression of ethylene-related ACO and ACS genes during flooding. In addition, correlation and RDA analysis showed that ARF1, ACO and ACS significantly promoted the expression of auxin, ACO and ACS enzyme activities, respectively (p < 0.05), while ADH, NR, AlaAT, ethylene emissions, Put, Spd, ACS and ACO were significantly correlated with ACS, ACO, and auxin-related gene expression (p < 0.05). Overall, ethylene emissions, ACS and ACO were identified as the main drivers of ethylene and auxin-related gene structure. ConclusionsThese results suggest that Spd regulated hormone concentrations, the content of Spd, Spm and Put, and related gene expression, in turn regulating physiological changes such as anaerobic enzyme activity, mitigating flooding stress in the roots and improving overall growth. Spd therefore has the potential to improve the adaptability of P. praecox to flooding stress.

Maternally-preset program of apoptosis and caspases involved in execution of the apoptosis at midblastula transition (MBT) but not before in &amp;lt;i&amp;gt;Xenopus laevis&amp;lt;/i&amp;gt; embryogenesis

To study gene control mechanisms in Xenopus embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into Xenopus fertilized eggs. The microinjection induced a large increase in SAMDC activity, exhaustion of the substrate SAM (S-adenosylmethionine), and execution of apoptosis at the stage called midblastula transition (MBT). By tracing GFP (green fluorescence protein)-marked apoptotic cells, we reached a conclusion that the apoptosis provides pre-blastula embryos with a fail-safe mechanism of early development. We analyzed caspase mRNAs and found that caspase-9 and -3 mRNAs are maternal mRNA and activation of caspase-9 is one of the key steps for the execution of the apoptosis. We also found that over- expression of caspase-8, and in addition p53, a tumor suppressor protein, also induces apoptosis at MBT, just like the overexpression of SAMDC and caspase-9 does. The apoptosis induced by p53 was suppressed by Xdm-2, a negative regulator of p53, and by a peptide inhibitor and a dominant-negative type mutant of caspase-9, but not by those of caspase-8. By contrast, apoptosis induced by SAMDC was suppressed by peptide inhibitors and dominant-negative mutants of both caspase-9 and caspase-8, but not by Xdm-2. Unlike caspase-9 mRNA, caspase-8 mRNA was not a maternal mRNA, but newly expressed during cleavage stage (pre-MBT stage) only in embryos overexpressed with SAMDC. In SAMDC-induced apoptotic embryos activities to process procaspase-8 and procaspase-9 appeared, whereas in p53-induced apoptotic embryos only activity to process procaspase-9 appeared. Thus, Xenopus embryos have at least two pathways to execute the maternal program of apoptosis: One induced by SAMDC overexpression through activation of caspase-9 and do novo expression of caspase-8 gene, and the other induced by p53 overexpression through activation of caspase-9 but not caspase-8. In Xenopus embryos, it has long been believed that zygotic genes are silent until MBT, but results obtained with caspase-8 may provide a novel example of gene expression before MBT.

Enhanced susceptibility of photosynthesis to low-temperature photoinhibition due to interruption of chill-induced increase of S-adenosylmethionine decarboxylase activity in leaves of spinach (Spinacia oleracea L.).

The possible involvement of polyamines in the chilling tolerance of spinach (Spinacia oleracea L.) was investigated focusing on photosynthesis. During chilling at 8/5C (day/night) for 6 d, S-adenosylmethionine decarboxylase (SAMDC) activity increased significantly in leaves in parallel with the increase in putrescine and spermidine (Spd) content in leaves and chloroplasts. Treatment of leaves with methylglyoxal-bis(guanylhydrazone) (MGBG), an SAMDC inhibitor, resulted in the deterioration of plant growth and photosynthesis under chilling conditions, which was reversed by the concomitant treatment with Spd through the roots. Plants treated with MGBG showed lower photochemical efficiency of PSII than either the control or plants treated with MGBG plus Spd during chilling and even after transfer to warm conditions, suggesting an increase of photoinhibition due to low Spd in chloroplasts. Indeed, MGBG-treated plants had much lower activities of thylakoid electron transport and enzymes in carbon metabolism as well as higher degrees of lipid peroxidation of thylakoid membranes compared to the control. These results indicate that the enhanced activity of SAMDC with a consequential rise of Spd in chloroplasts is crucial for the cold acclimation of the photosynthetic apparatus in spinach leaves.

The early increase of S-adenosylmethionine decarboxylase activity is essential for the normal germination and tube growth in tomato ( Lycopersicon esculentum Mill.) pollen

The effect of polyamine biosynthesis inhibitors on pollen germination and tube growth and putrescine (Put), spermidine (Spd) and spermine (Spm) levels in germinating tomato ( Lycopersicon esculentum Mill.) pollen was investigated. Pollen germination was initiated 20 min after incubation at 25 °C and completed within 2 h. The activity of arginine decarboxylase (ADC), an enzyme catalyzing Put synthesis, exhibited a marked increase during the initial 2 h and remained high thereafter. The activity of S-adenosylmethionine decarboxylase (SAMDC), an essential enzyme in the synthesis of Spd and Spm, increased transiently during the first hour of incubation. Consequently, the germinating pollen showed elevated ratios of Spd+Spm to Put during the first hour in all of the free, conjugated and bound forms. The addition to the germination medium of either methylglyoxal-bis(guanylhydrazone) (MGBG) or cyclohexylamine, inhibitors of SAMDC and spermidine synthase, respectively, caused a marked decrease of Spd and Spm contents, together with ratios of Spd+Spm to Put, and resulted in inhibition of pollen germination and tube growth. Simultaneous addition of Spd or Spm reversed the adverse effect of MGBG. However, Put failed to reverse the inhibitory effect of d-arginine, a competitive inhibitor of ADC. MGBG treatment 2 h after incubation was still inhibitory to tube growth, which was partially reversed by Spd. The results suggest that the increased activity of SAMDC and the resultant elevation of Spd and Spm contents during the early pollen germination phase are essential for the occurrence of normal germination and tube growth in tomato pollen.

Influence of physical exercise on polyamine synthesis in the rat skeletal muscle.

Physical exercise and testosterone administration result in a series of adaptive anabolic phenomena in the skeletal muscle. The role of polyamines in these processes has been poorly explored. We measured the activities of polyamine-synthesising enzymes, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) and polyamine content in skeletal muscle of male rats exposed to endurance or resistance exercise, or a single testosterone treatment. Soleus muscle (consisting mainly of slow-twitching oxidative fibres-STO) and extensor digitorum longus (mainly fast-twitching glycolytic muscle fibres-FTG) were analysed for polyamine content by HPLC, and ODC and SAMDC activity. Both endurance and resistance exercise induced a threefold increase in endogenous testosterone production. Two hours after exercise, ODC was increased in STO fibres, returning to baseline after 24 h; in FTG fibres the increase was less prominent. An increase in SAMDC activity occurred in a more sustained manner, with its peak 8 h after exercise. Polyamines were subsequently accumulated in both skeletal muscle fibres, with a rise in putrescine concentration after 2 h, and a fall corresponding to conversion of putrescine to spermidine and spermine by SAMDC. Single dose of 17alpha-methyltestosterone resulted in a similar increase in polyamine-synthesising enzyme activities and polyamine concentrations in the skeletal muscle. Polyamine accumulation in the skeletal muscle after physical exercise is likely to occur secondary to testosterone production. Polyamines are apparently involved in the oxidative, but not in glycolytic processes related to muscle adaptation to exercise.

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in early Xenopus embryos induces cell dissociation and inhibits transition from the blastula to gastrula stage.

Xenopus early embryos contain relatively low levels of S-adenosyl-methionine decarboxylase (SAMDC) and its mRNA. When SAMDC mRNA was injected into Xenopus embryos, it was preserved until the blastula stage and induced a large increase in SAMDC activity. The SAMDC-overexpressed embryos developed normally until the blastula stage but at the early gastrula stage cells which received the mRNA, dissociated autonomously and stopped synthesizing protein. In a hypotonic medium, the dissociated cells, and hence whole embryos, autolyzed. However, in isotonic media dissociated cells did not autolyze, although they did not divide and their DNA and RNA synthesis activity was greatly inhibited. The effects of SAMDC overexpression were abolished by coinjection of ethylglyoxal-bis(guanylhydrazone) (EGBG), a specific inhibitor of SAMDC. In SAMDC-overexpressed embryos the level of putrescine decreased and that of spermidine increased, though to limited extents, resulting in a considerable decrease in the putrescine/spermidine ratio. However, direct injection of spermidine did not mimic the effect of SAMDC overexpression, and putrescine coinjected with SAMDC mRNA to maintain the normal putrescine/spermidine ratio did not rescue the embryos. Conversely, the level of S-adenosylmethionine (SAM) greatly decreased and coinjection of SAM, which restored the level of SAM, rescued the embryos. We concluded that in SAMDC-overexpressed embryos a SAM-deficient state was induced and this caused cell dissociation and inhibition of transition from the blastula to gastrula stage. We suggest that the SAM-deficient embryos obtained in the present study provide a unique system for studying the cellular control mechanism underlying the blastula-gastrula transition.

S-adenosylmethionine decarboxylase in human brain. Regional distribution and influence of aging

Recent experimental animal studies have implicated brain polyamines as having roles in both brain development and human brain neurodegenerative conditions. In order to provide baseline information, in normal human brain, on one of the key polyamine synthesising enzymes, S-adenosylmethionine decarboxylase (SAMDC), we examined the sensitivity of this enzyme to various cofactors/inhibitors, its regional distribution, and influence of aging in neurologically normal autopsied human brain. SAMDC in normal human brain is similar to that reported in other mammalian cells with regard to substrate affinity ( K m = 39 μM), marked sensitivity to putrescine activation (+600%), inhibition (methylglyoxalbisguanidine and MDL 73811), and pH optimum (7.2). There was an uneven distribution of enzyme activity in human brain, and of the 12 brain regions examined, the highest activity was observed in occipital, parietal, frontal and temporal cortices (36–58 pmol/h/mg protein); intermediate activity in cerebellar and insular cortex, pulvinar thalamus, caudate and putamen (12–27 pmol/h/mg protein); and lowest activity in medial-dorsal thalamus, lateral globus pallidus and white matter (< 11 pmol/h/mg protein). The influence of aging (1 day to 103 years) on SAMDC activity in occipital cortex, the region showing the highest activity in human brain ( n = 59) was also determined. Enzyme activity increased by approximately 600% from age 6 months to near maximal levels at age 10 years, then remained generally unchanged up to 103 years. Since SAMDC is a key regulatory enzyme in the synthesis of spermidine and spermine, the marked increase in SAMDC activity in the neonate and the sustained high enzyme levels throughout adulthood, imply a role for these polyamines in both development and mature brain function.

Structure and regulation of mammalian S-adenosylmethionine decarboxylase.

In order to understand the structure and regulation of S-adenosylmethionine decarboxylase, cDNA clones encoding this enzyme have been isolated from rat prostate and human fibroblast cDNA libraries. The authenticity of the cDNAs was verified by: (a) transfecting the Chinese hamster ovary cells with the human cDNA in the pcD vector which resulted in a transient 10-20-fold increase in S-adenosylmethionine decarboxylase activity in recipient cells; and (b) translating the mRNA formed by transcription of the cDNA insert in a reticulocyte lysate and recording an increase in S-adenosylmethionine decarboxylase activity. The amino acid sequences deduced from the cDNAs indicate that the human proenzyme for this protein contains 334 amino acids and has a molecular weight of 38,331 whereas the rat proenzyme contains 333 amino acid residues. The human and rat enzymes are very similar having only 11 amino acid differences and the cDNAs are also closely related showing over 90% homology in the 1617-nucleotide overlap which was sequenced. A further indication of the highly conserved nature of mammalian S-adenosylmethionine decarboxylases is that the amino acid sequences deduced from the human and the rat cDNAs contained peptide sequences identical to those previously reported for the purified bovine enzyme. In vitro transcription/translation experiments showed that the proenzyme is converted to two polypeptides of molecular weights about 32,000 and 6,000 in a processing reaction which generates the prosthetic pyruvate group and that the final enzyme contains both polypeptides. Two forms of S-adenosylmethionine decarboxylase mRNA (2.1 and about 3.4-3.6 kilobases) are present in human and rodent tissues and may originate from the utilization of two different polyadenylation signals. Southern blots of rat genomic DNA indicated that the S-adenosylmethionine decarboxylase gene belongs to a multigene family. Depletion of cellular polyamines by inhibitors or ornithine decarboxylase or the aminopropyltransferases led to an increase in the content of S-adenosylmethionine decarboxylase protein and mRNA, but the elevation in the mRNA was not sufficient to account for all of the change in the enzyme level, particularly in cells in which spermine was depleted.

Polyamine depletion increases cellular ribonucleotide levels.

Depletion of the putrescine and spermidine content of Ehrlich ascites tumor cells by alpha-difluoromethylornithine (DFMO) treatment results in at least a 1 500-fold increase in the decarboxylated S-adenosylmethionine (deSAM) content. The accumulation of this adenine nucleoside occurs because of the absence of putrescine and spermidine to act as aminopropyl group acceptors in the spermidine and spermine synthase reactions and because of an increase in S-adenosylmethionine decarboxylase activity. The fact that the synthesis of deSAM continues in DFMO-treated cells makes the pathway an adenine trap. This prompted a study of the adenine nucleotide pools. High-performance liquid chromatographic analysis showed that the total adenine nucleotide pool increased, rather than decreased, as a result of DFMO treatment; the major contributors to the increase being ATP and ADP, which increased 2.6 and 1.9 times, respectively. The cellular content of other ribonucleotides increased as well, particularly that of UTP and CTP. When putrescine was added together with DFMO, the increases in cellular ribonucleotide contents were prevented, showing that they were indeed caused by polyamine depletion.

Effect of 1,25-dihydroxyvitamin D on S-adenosylmethionine decarboxylase in chick intestine

The effect of 1,25-dihydroxyvitamin D on the activity of S-adenosylmethionine decarboxylase in the duodenal mucosa of vitamin D-deficient chicks was investigated. Enzyme activity increased dose-dependently in a biphasic manner with maximal responses at 1 and 6 h, due to an increase in V max in both cases. A second dose of 1,25-dihydroxyvitamin D, administered 6 h after the first, resulted in a significant increase in activity 1 h later, confirming the rapidity of the response. This early response was not seen with ornithine decarboxylase. The increase in S-adenosylmethionine decarboxylase activity particularly at 6 h may be due to a rise in cytosolic calcium, since hydrocortisone, an inhibitor of 1,25-dihydroxyvitamin D-stimulated calcium absorption, attenuates this enzyme's activity. Inhibitors of polyamine biosynthesis such as DL-α-difluoromethyl ornithine and methylglyoxal bis(guanylhydrazone) had no effect on calcium absorption, but the significance of this in evaluating the importance of 1,25-dihydroxyvitamin D stimulation of S-adenosylmethionine decarboxylase activity needs further investigation.

Polyamine metabolism in rat lungs with oxygen toxicity

Ornithine decarboxylase activity increases 2-fold above control after 1 day and 25-fold after 3 days of exposure to 0.85 atm oxygen. Putrescine content nearly doubled by 72 hours which may reflect increased activity of ornithine decarboxylase. Spermidine and spermine content did not increase until after 3 days of exposure which was consistent with the delayed increase of S-adenosylmethionine decarboxylase activity. The results suggest that antimetabolites of polyamine metabolism may be useful to suppress excessive cellular proliferation in the lung after acute lung injury.

Arginase, ornithine decarboxylase and S-adenosylmethionine decarboxylase in chicken brain and retina

1. 1. Arginase, ornithine decarboxylase and S-adenosylmethionine decarboxylase are active in both retina and brain. Activity is higher in cerebellum than in the cerebral hemispheres and optical lobes. 2. 2. Arginase and ornithine decarboxylase are very active in the retina of very young chicks, while S-adenosylmethionine decarboxylase is poorly active. By contrast, S-adenosylmethionine decarboxylase is much more active in brain. 3. 3. The pattern of activity during development is different; only ornithine decarboxylase is very active during embryonal life; S-adenosylmethionine decarboxylase, at all events in brain, is more active in adult life. 4. 4. Ornithine decarboxylase is inhibited in vitro by α-difluoromethylornithine, but not in vivo. Diaminopropane inhibits brain ornithine decarboxylase, but does not induce an ornithine decarboxylase-antizyme. 5. 5. Methylglyoxal bis(guanylhydrazone) promotes an increase of S-adenosylmethionine decarboxylase activity in both the brain and the retina in vivo.

Effect of methionine deprivation on S-adenosylmethionine decarboxylase of tumour cells

Transference of Walker carcinoma and TLX5 lymphoma from normal L-methionine-containing medium to medium containing limiting amounts of L-methionine, or L-homocysteine only, caused a 2-fold increase of S-adenosylmethionine decarboxylase activity. Kinetic analysis showed an increase in the V value of the enzyme from 22 to 53 pmol/min per mg protein in media containing only 0.1 mM L-homocysteine, without any alteration in the K m value (0.1 mM). The increase in enzyme activity does not result from (a) a reduction of the intracellular level of S-adenosylmethionine, since cycloleucine, an inhibitor of methionine adenosyltransferase, had no effect on enzyme activity; (b) an increase in intracellular adenosine 3′,5′ monophosphate (cyclic AMP), since high extracellular concentrations of N 6-monobutyryl cyclic AMP had no effect on enzyme activity; (c) an alteration of polyamine levels, since addition of micromolar concentrations of exogenous putrescine, spermidine and spermine did not prevent the induction of S-adenosylmethionine decarboxylase activity in methionine-free media containing 0.1 mM L-homocysteine. The increased enzyme activity appears to be mainly due to enhanced stabilization, since the half-life was increased from 2.45 to 5.0 h in media containing only 0.1 mM L-homocysteine. Induction of enzyme activity is specific to the removal of L-methionine, since no increase occured in the absence of L-serine or L-glycine, or both, or by reduction of the serum concentrations in the medium.

Effect of Malnutrition and Rehabilitation on the Metabolism of Polyamines in Rat Liver

In adult male rats the activity of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMD) and the concentration of putrescine and spermidine increased markedly 3 to 4 weeks after feeding the animals a 6% casein diet. Incorporation of [14C]orotic acid into nuclear RNA also increased during this period of time. In contrast, spermine concentration remained unchanged. During the fifth week of protein restriction enzyme activities, putrescine and spermidine concentrations and orotic acid incorporation returned to control levels. After the restricted rats were put on a control diet there was a rapid increase above control levels in ODC activity and a proportionally smaller increase in SAMD activity. Putrescine and spermidine concentration also increase above control levels. Both enzyme activities and polyamine concentrations were within normal levels 96 hours after refeeding. Spermine values were unmodified by refeeding. After a transient rise 12 hours after refeeding, incorporation of labeled orotic acid into RNA decreased significantly for the rest of the experiment.

Ornithine decarboxylase in rat small intestine: stimulation with food or insulin.

Ornithine decarboxylase in the small intestine of starved rats was stimulated 3- to 10-fold by refeeding or administration of insulin. A peak is observed 3-5 h following treatment after which the enzyme activity rapidly declines. The rise in ornithine decarboxylase is reduced by actinomycin D or cycloheximide. The increase in enzyme activity occurs mainly in the duodenum and jejunum with less than a twofold change being observed in the ileum. A small (twofold) increase in S-adenosylmethionine decarboxylase activity in the small intestine was observed after food, but there was no change in diamine oxidase activity. Whereas pentagastrin and metiamide administration markedly stimulated histidine decarbosylase in the gastric mucosa, no consistent effect of these agents on ornithine decarboxylase in the small intestine was observed. The similarities and differences between histidine decarboxylase and ornithine decarboxylase are discussed.