Polyamine Metabolism and Osmotic Stress : I. Relation to Protoplast Viability

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine, and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, andVigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.     

Thermal inactivation of a Citrobacter ribonuclease: Influence of polyamines and ionic strength

The thermal inactivation of a Citrobacter sp. ribonuclease (RNase) is subject to control by a number of factors. Low concentrations of naturally occurring polyamines such as spermidine and spermine, and certain analogs of these compounds, protect the enzyme from inactivation. Changes in ionic strength cause wide variations in the rate at which enzyme activity is lost. Additionally, depending on the type of ion added to the reaction mixture, the rate constant for enzyme inactivation-may either increase or decrease as the ionic strength is raised. Thermodynamic parameters were determined under a variety of experimental conditions for the thermal inactivation of this RNase. It was found in all of these cases that the entropy of activation is large and negative, implying that a gross change in enzyme conformation is not taking place. The concentration and identity of ions present and the amount of polyamine available to interact with this RNase determines the rate of loss, by thermal inactivation, of enzyme activity in this in vitro system. These factors therefore constitute a system whereby substrate hydrolysis may be controlled with time.     

Age dependency of the metabolic conversion of polyamines into amino acids in IMR-90 human embryonic lung diploid fibroblasts

When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000-dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000-dalton protein. We have investigated these two polyamine-associated biochemical events in IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL). We found that (1) the metabolic labeling of the 18,000-dalton protein was about two-fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six-fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4-fold higher in the young cells as compared to the old cells, our data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR-90 cells was about eight-fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000-dalton protein were also observed in cell strains derived from patients with premature aging disease.     

Polyamine levels as related to growth,differentiation and senescence in protoplast-derived cultures of Vigna aconitifolia and Avena sativa

We have previously reported that aseptically cultured mesophyll protoplasts of Vigna divide rapidly and regenerate into complete plants, while mesophyll protoplasts of Avena divide only sporadically and senesce rapidly after isolation. We measured polyamine titers in such cultures of Vigna and Avena, to study possible correlations between polyamines and cellular behavior. We also deliberately altered polyamine titer by the use of selective inhibitors of polyamine biosynthesis, noting the effects on internal polyamine titer, cell division activity and regenerative events.In Vigna cultures, levels of free and bound putrescine and spermidine increased dramatically as cell division and differentiation progressed. The increase in bound polyamines was largest in embryoid-forming callus tissue while free polyamine titer was highest in root-forming callus. In Avena cultures, the levels of total polyamines decreased as the protoplast senesced. The presence of the inhibitors -difluoromethyl-arginine (specific inhibitor of arginine decarboxylase) and dicyclohexylamine (inhibitor of spermidine synthase) reduced cell division and organogenesis in Vigna cultures. Addition of low concentration of polyamines to such cultures containing inhibitors or removal of inhibitors from the culture medium restored the progress of growth and differentiation with concomitant increase in polyamine levels.     

Polyamine Synthesis by the Mermithid Nematode Romanomermis culicivorax

The polyamine and amino acid composition of the mermithid nematode, Romanomermis culicivorax, and its host, Aedes aegypti, was determined. Putrescine, spermidine, spermine, cadaverine and two acetylated spermidine derivatives were present in parasitic juveniles, newly-emerged post-parasites, and eggs of R. culicivorax. Whole insect homogenates of fourth-instar A. aegypti contained the same array of polyamines, except that the putrescine:spermidine ratio was the inverse of that in parasitic R. culicivorax. Polyamines and amino acids were in greater concentrations in the nematode eggs than in other developmental stages investigated. Both the host and nematode possess the biosynthetic capacity for polyamine biosynthesis, as evidenced by measurable activities of ornithine decarboxylase in the host''s tissues and the nematode''s free-living stages.     

Biosynthesis of long-chain polyamines by crenarchaeal polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum

Polyamines are ubiquitously present in all organisms. In addition to the common polyamines, thermophilic archaea synthesize long-chain polyamines. In the present study polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum were cloned and their substrate specificity was analyzed. The polyamine synthase HbSpeE II from H. butylicus synthesized long-chain polyamines with high activity using the same mechanism that is used by a wide range of organisms to synthesize common polyamines, in which the aminopropyl residue derives from decarboxylated S-adenosylmethionine. This is the first polyamine synthase described that synthesizes a polyamine longer than a tetramine with high activity.     

The role of polyamines in protein-dependent hypoxic tolerance of Drosophila

Background

Chronic hypoxia is a major component of ischemic diseases such as stroke or myocardial infarction. Drosophila is more tolerant to hypoxia than most mammalian species. It is considered as a useful model organism to identify new mechanisms of hypoxic tolerance. The hypoxic tolerance of flies has previously been reported to be enhanced by low protein diets. This study analyses the mechanisms involved.

Results

Feeding adult Drosophila on a yeast diet dramatically reduced their longevities under chronic hypoxic conditions (5% O₂). Mean and maximum longevities became close to the values observed for starving flies. The action of dietary yeast was mimicked by a whole casein hydrolysate and by anyone of the 20 natural amino acids that compose proteins. It was mimicked by amino acid intermediates of the urea cycle such as L-citrulline and L-ornithine, and by polyamines (putrescine, spermidine and spermine). α-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, partially protected hypoxic flies from amino acid toxicity but not from polyamine toxicity. N¹-guanyl-1,7 diaminoheptane, a specific inhibitor of eIF5A hypusination, partially relieved the toxicities of both amino acids and polyamines.

Conclusion

Dietary amino acids reduced the longevity of chronically hypoxic flies fed on a sucrose diet. Pharmacological evidence suggests that the synthesis of polyamines and the hypusination of eIF5A contributed to the life-shortening effect of dietary amino acids.     

Comparative role of polyamines division and plastid differentiation of Euglena gracilis

Regulation of polyamine biosynthesis during growth and differentation of Euglena gracilis was investigated. Increased activity of l-ornithine decarboxylase (EC 4.1.1.17), the enzyme which catalyzes the initial step in polyamine synthesis in Euglena, and accumulation of polyamines were observed prior to DNA replication in synchronous cultures of heterotropically or photoautotrophically grown cells. In photoatotrophic cells three maxima of polyamine synthesis were observed during the light period of the cell cycle. The transition from quiescence of active growth was accompanied in heterotrophic Euglena by a very large stimulation of ornithine decaboxylase activity and polyamine synthesis; the decrease in growth potential of these cells was correlated with a decrease in polyamine levels. In contrast, differentiation of Euglena, i.e., a shift from heterotrophic to photoautotrophic mode of living in the absence of division, led only to a minor stimulation of polyamine biosynthesis. α-Methylornithine, an inhibitor of ornithine decarboxylase, blocked the growth of heterotrophic Euglena, and depletion of intracellular polyamines decreased the differentiation rate. Both events could be reversed only by addition of putrescine to the growth medium. This study suggests that Euglena requires a minimal intracellular level of polyamines to grow and differentiate under optimal conditions. This requirement seems to be more stringent for cell division.     

Exogenous calcium affects nitrogen metabolism in root-zone hypoxia-stressed muskmelon roots and enhances short-term hypoxia tolerance

We investigated the effects of short-term root-zone hypoxic stress and exogenous calcium application or deficiency in an anoxic nutrient solution on nitrogen metabolism in the roots of the muskmelon cultivar Xiyu No. 1. Seedlings grown in the nutrient solution under hypoxic stress for 6 d displayed significantly reduced plant growth and soluble protein concentrations. However, NO₃⁻ uptake rate and activities of nitrate reductase and glutamate synthase were significantly increased. We also found higher amounts of nitrate, ammonium, amino acids, heat-stable proteins, polyamines, H₂O₂, as well as higher polyamine oxidase activity in the roots. In comparison to the reactions seen under hypoxic stress, exogenous calcium application led to a marked increase in plant weights, photosynthesis parameters, NO₃⁻ uptake rate and contents of nitrate, ammonium, amino acids (e.g., glutamic acid, proline, glycine, cystine, γ-aminobutyric acid), soluble and heat-stable proteins, free spermine, and insoluble bound polyamines. Meanwhile, exogenous calcium application resulted in significantly increased activities for nitrate reductase, glutamine synthetase, and glutamate synthase but decreased activities for diamine and polyamine oxidase, as well as lower H₂O₂ content in roots during exposure to hypoxia. However, calcium deficiency in the nutrient solution decreased plant weight, photosynthesis parameters, NO₃⁻ reduction, amino acids (e.g., alanine, aspartic acid, glutamic acid, γ-aminobutyric acid), protein, all polyamines except for free putrescine, and the activities of glutamate synthase and glutamine synthetase. Additionally, there was an increase in the NO₃⁻ uptake rate, polyamine oxidase activity and H₂O₂ contents under hypoxia-Ca. Simultaneously, exogenous calcium had little effect on nitrate absorption and transformation, photosynthetic parameters, and plant growth under normoxic conditions. These results suggest that calcium confers short-term hypoxia tolerance in muskmelon, most likely by promoting nitrate uptake and accelerating its transformation into amino acids, heat-stable proteins or polyamines, as well as by decreasing polyamine degradation in muskmelon seedlings.     

Micro determination of polyamines with snake venom oxidase.

An accurate micromethod suitable for the assay of polyamines in concentrations of 2 to 30 nmol is described. It is based on the oxidation of polyamines by purified fractions of crude l-amino acid oxidase from Russell's viper venom. By a combination of two enzyme fractions, one which oxidizes polyamines and amino acids (AAP) and another which oxidizes only amino acids (AA), the technique can accurately determine polyamine concentrations in extracts of sera which may not be free of amino acids. Experiments described show 90 to 100% recovery of added polyamines in the presence of varying amounts of amino acids. Polyamines added to serum also showed recoveries ranging from 96 to 98%. The enzymes do not oxidize histamine and epinephrine and are very stable. The method does not require sophisticated equipment and is suitable for screening of large number of clinical samples to assess the importance of polyamines as a diagnostic test or their prognostic value in diseases like cancer.     

Rescue of the fission yeast snRNA synthesis mutant snm1 by overexpression of the double-strand-specific Pac1 ribonuclease

The Schizosaccharomyces pombe temperature-sensitive mutant snm1 maintains reduced steady-state quantities of the spliceosomal small nuclear RNAs (snRNAs) and the RNA subunit of the tRNA processing enzyme RNase P. We report here the isolation of the pac1 ⁺ gene as a multi-copy suppressor of snm1. The pac1 ⁺ gene was previously identified as a suppressor of the ran1 mutant and by its ability to cause sterility when overexpressed. The pac1 ⁺ gene encodes a double-strand-specific ribonuclease that is similar to RNase III, an RNA processing and turnover enzyme in Escherichia coli. To investigate the essential structural features of the Pac1 RNase, we altered the pac1 ⁺ gene by deletion and point mutation and tested the mutant constructs for their ability to complement the snm1 and ran1 mutants and to cause sterility. These experiments identified four essential amino acids in the Pac1 sequence: glycine 178, glutamic acid 251, and valines 346 and 347. These amino acids are conserved in all RNase III-like proteins. The glycine and glutamic acid residues were previously identified as essential for E. coli RNase III activity. The valines are conserved in an element found in a family of double-stranded RNA binding proteins. Our results support the hypothesis that the Pac1 RNase is an RNase III homolog and suggest a role for the Pac1 RNase in snRNA metabolism.     

Regulations of thyroid decarboxylase by the polyamines Induction of a protein inhibitor of ornithine decarboxylase by the end-products of the reaction

When spermdine, putrescine or 1,3-diaminopropane was injected (12.5 μmol/100 g body weight) into rats i h before thyrotropin, ornithine decarboxylase activity was increased by 75–150% over control levels. However, when 75 μmol polyamine/100 g body weight was injected, thyrotropin-activated activity was inhibited by 70–95%. Multiple polyamine injections inhibited goitrogen-induced activity and gland weight increase by approx. 35%.The polyamines also inhibited thyrotrophin-activated rat thyroid ornithine decarboxylase in vitro in a dose-related fashion, with 50% inhibition occurring at 2–5 · 10⁻⁴ M. The inhibition was not due to a direct effect on the enzyme. No stimulation was seen with low concentration of polyamine. The polyamines had no effect on in vitro thyroid protein/RNA synthesis or glucose oxidation but had a biphasic effect on plasma membrane adenylate cyclase activity.A protein inhibitor to thyroid ornithine decarboxylase was generated in vivo by multiple injections of the polyamines into rats, and in vitro by incubating bovine thyroid slices with 2–10 mM polyamine. The inhibitor was non-dialyzable, destroyed by boiling, and its formation was blocked in a dose-related fashion by cycloheximide.We conclude that: (1) thyroid ornithine decarboxylase is subject not only to positive control, but is also negatively regulated by its end-products, the polyamines, which induce a protein inhibitor to ornithine decarboxylase; (2) since gland growth is also inhibited under these conditions, the polyamine effect on thyroid ornithine decarboxylase may be biologically significant.     

Regulation of thyroid ornithine decarboxylase by the polyamines. Induction of a protein inhibitor of ornithine decarboxylase by the end-products of the reaction

When spermidine, putrescine or 1,3-diaminopropane was injected (12.5 mumol/100 g body weight) into rats 1 h before thyrotropin, ornithine decarboxylase activity was increased by 75--150% over control levels. However, when greater than or equal to 75 mumol polyamine/100 g body weight was injected, thyrotropin-activated activity was inhibited by 70--95%. Multiple polyamine injections inhibited goitrogen-induced activity and gland weight increase by approx 35%. The polyamines also inhibited thyrotropin-activated rat thyroid ornithine decarboxylase in vitro in a dose-related fashion, with 50% inhibition occurring at 2--5 . 10(-4)M. The inhibition was not due to a direct effect on the enzyme. No stimulation was seen with low concentrations of polyamine. The polyamines had no effect on in vitro thyroid protein/RNA synthesis or glucose oxidation but had a biphasic effect on plasma membrane adenylate cyclase activity. A protein inhibitor to thyroid ornithine decarboxylase was generated in vivo by multiple injections of the polyamines into rats and in vitro by incubating bovine thyroid slices with 2--10 mM polyamine. The inhibitor was non-dialyzable, destroyed by boiling, and its formation was blocked in a dose-related fashion by cycloheximide. We conclude that: (1) thyroid ornithine decarboxylase is subject not only to positive control, but is also negatively regulated by its end-products, the polyamines, which induce a protein inhibitor to ornithine decarboxylase; (2) since gland growth is also inhibited under these conditions, the polyamine effect on thyroid ornithine decarboxylase may be biologically significant.     

Polyamine auxotrophs of Saccharomyces cerevisiae.

Strains of yeast have been constructed that are unable to synthesize ornithine and are thereby deficient in polyamine biosynthesis. These strains were used to develop a protocol for isolation of mutants blocked directly in polyamine synthesis. There were seven mutants isolated that lack ornithine decarboxylase activity; these strains exhibited greatly decreased pool levels of putrescine, spermidine, and spermine when grown in the absence of polyamines. Three of the mutants lack S-adenosylmethionine decarboxylase activity; polyamine limitation of a representative mutant resulted in an accumulation of putrescine and a decrease in spermidine and spermine. When the mutants were cultured in the absence of polyamines, a continuously declining growth rate was observed.     

Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N ¹-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis.     

Agp2, a Member of the Yeast Amino Acid Permease Family,Positively Regulates Polyamine Transport at the Transcriptional Level

Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3Δ nor sam3Δ single mutant is defective in polyamine transport, while the dur3Δ sam3Δ double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2Δ mutant. Studies of Agp2 localization indicate that in the double mutant dur3Δ sam3Δ, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport.     

Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus. The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60°C. The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity. The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP. When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition. The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity. Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC. Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T. thermophilus, and in a decrease of the specific activity of ornithine decarboxylase. The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az). The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E. coli antizyme. The antizyme of E. coli inhibited the ODC of T. thermophilus, and vice versa. The fragment of amino acids 56-292 of the E. coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E. coli or T. thermophilus.     

Acetylation of spermidine and spermine by rat liver and kidney chromatin

The in vitro enzymatic acetylation of the polyamines, spermidine and spermine, is described. The reaction is catalyzed by chromatin preparations from rat liver and kidney and is dependent on acetyl-CoA. Spermidine, spermine, and putrescine are each converted to the corresponding monoacetyl derivatives. s_0.5 values of 0.5 ± 0.1, 1.0 ± 0.1, and 2.6 ± 0.7 mm (mean ± standard deviation) were obtained for spermidine, spermine, and putrescine, respectively. These values for s_0.5 are similar to the concentrations of polyamines reported for tissues, and therefore, suggest the occurrence of polyamine acetylation in vivo. Evidence is also presented for the metabolism of acetylated polyamines by the 100,000g supernatant fraction of rat liver. The physiological function of polyamine acetylation is unknown, but the possibility of an effect on the association of polyamines with nucleic acids is discussed.     

Excretion of Endogenous Cadaverine Leads to a Decrease in Porin-Mediated Outer Membrane Permeability

The permeability of the outer membrane of Escherichia coli to hydrophilic compounds is controlled by porin channels. Electrophysiological experiments showed that polyamines inhibit ionic flux through cationic porins when applied to either side of the membrane. Externally added polyamines, such as cadaverine, decrease porin-mediated fluxes of β-lactam antibiotics in live cells. Here we tested the effects of endogenously expressed cadaverine on the rate of permeation of cephaloridine through porins, by manipulating in a pH-independent way the expression of the cadBA operon, which encodes proteins involved in the decarboxylation of lysine to cadaverine and in cadaverine excretion. We report that increased levels of excreted cadaverine correlate with a decreased outer membrane permeability to cephaloridine, without any change in porin expression. Cadaverine appears to promote a sustained inhibition of porins, since the effect remains even after removal of the exogenously added or excreted polyamine. The cadaverine-induced inhibition is sufficient to provide cells with some resistance to ampicillin but not to hydrophobic antibiotics. Finally, the mere expression of cadC, in the absence of cadaverine production, leads to a reduction in the amounts of OmpF and OmpC proteins, which suggests a novel mechanism for the environmental control of porin expression. The results presented here support the notion that polyamines can act as endogenous modulators of outer membrane permeability, possibly as part of an adaptive response to acidic conditions.