Polyamine levels and the activity of their biosynthetic enzymes in human erythrocytes infected with the malarial parasite, Plasmodium falciparum.

Human erythrocytes contain only trace amounts of polyamines and lack active polyamine biosynthetic enzymes. A remarkable increase in polyamine content, and in the activity of ornithine and S-adenosyl-L-methionine decarboxylases, is noted in synchronous cultures of the malarial parasite, Plasmodium falciparum. Polyamine biosynthesis reached peak values during the early trophozoite stage, whereas nucleic acid and protein synthesis occurred later in mature trophozoites. DL-alpha-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, did not interfere with merozoite invasion and with ring-form development, but prevented the transformation of trophozoites to schizonts. Concomitantly, the synthesis of proteins and nucleic acids was significantly inhibited. These inhibitory effects could be readily reversed by the diamine putrescine. Macromolecular synthesis and schizogony were normal when 5-10 mM-DL-alpha-difluoromethylornithine and 0.1 mM-putrescine were added to the cultures simultaneously.     

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S₂₀/L₂₆ and L₃₄, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.     

Effect of 1,3-diaminopropan-2-o1, an inhibitor of ornithine decarboxylase, on the metabolic response of liver to insulin

The effect of diaminopropanol, an inhibitor of polyamine synthesis, on the metabolic response of liver to insulin was studied in streptozotocin-diabetic rats. Insulin elicited a prompt and very marked increase in ornithine and S-adenosylmethionine decarboxylase activities and in putrescine concentration. Pretreatment of rats with diaminopropanol prevented the increase in the decarboxylases and resulted in decreased spermidine and spermine content of liver. The insulin-induced increase in glycogen content was depressed by 50% and the increase in the rate of lipogenesis in vivo was completely prevented by prior injection of diaminopropanol. These studies implicate altered polyamine metabolism in the metabolic response of liver of streptozotocin-diabetic rats to insulin.     

Response of enzymes involved in the metabolism of polyamines to phytohaemagglutinin-induced activation of human lymphocytes.

The stimulation of lymphocyte ornithine decarboxylase and adenosylmethionine decarboxylase produced by phytohaemagglutinin was accompanied by an equally marked, but delayed, stimulation of spermidine synthase, which is not commonly considered as an inducible enzyme. In contrast with the marked stimulation of these biosynthetic enzymes, less marked changes were observed in the biodegradative enzymes of polyamines in response to phytohaemagglutinin. Diamine oxidase activity was undetectable during all stages of the transformation. The activity of polyamine oxidase remained either constant or was slightly decreased several days after addition of the mitogen. The activity of polyamine acetylase (employing all the natural polyamines as substrates) distinctly increased both in the cytosolic and crude nuclear preparations of the cells during later stages of mitogen activation. Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, although powerfully inhibiting ornithine decarboxylase, produced a gradual enhancement of adenosylmethionine decarboxylase activity during lymphocyte activation, without influencing the activities of the two propylamine transferases (spermidine synthase and spermine synthase).     

Stimulation of ornithine decarboxylase activity and inhibition of S-adenosyl-l-methionine decarboxylase activity in leukaemic mice by methylglyoxal bis(guanylhydrazone) (Short Communication)

Administration of methylglyoxal bis(guanylhydrazone) to leukaemic mice results in an early depression followed by a marked elevation of S-adenosyl-l-methionine decarboxylase activity. Further, there is an early prolonged increase in the activity of ornithine decarboxylase, the initial enzyme in the polyamine biosynthetic pathway. Because of the profound effects of methylglyoxal bis(guanylhydrazone) in vivo on the polyamine biosynthetic pathway, the drug can no longer be considered a specific inhibitor of spermidine synthesis.     

Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally.     

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.     

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.     

The effect of salt stress on polyamine biosynthesis and content in mung bean plants and in halophytes

The activity of L-arginine decarboxylase (EC 4.1.1.19) and L-ornithine decarboxylase (EC 4.1.1.17), polyamine content, and incorporation of arginine and ornithine into polyamines, were determined in mung bean [Vigna radiata (L.) Wilczek] plants subjected to salt (hypertonic) stress (NaCl at 0.51–2.27 MPa). Changes in enzyme activity in response to hypotonic stress were determined as well in several halophytes [Pulicaria undulata (L.), Kostei, Salsola rosmarinus (Ehr.) Solms-Laub, Mesembryanthemum forskahlei Hochst, and Atriplex halimus L.]. NaCl stress, possibly combined with other types of stress that accompanied the experimental conditions, resulted in organ-specific changes in polyamine biosynthesis and content in mung bean plants. The activity of both enzymes was inhibited in salt-stressed leaves. In roots, however, NaCl induced a 2 to 8-fold increase in ornithine decarboxylase activity. Promotion of ornithine decarboxylase in roots could be detected already 2 h after exposure of excised roots to NaCl, and iso-osmotic concentrations of NaCl and KCl resulted in similar changes in the activity of both enzymes. Putrescine level in shoots of salt-stressed mung bean plants increased considerably, but its level in roots decreased. The effect of NaCl stress on spermidine content was similar, but generally more moderate, resulting in an increased putrescine/spermidine ratio in salt-stressed plants. Exposure of plants to NaCl resulted also in organ-specific changes in the incorporation of both arginine and ornithine into putrescine: incorporation was inhibited in leaf discs but promoted in excised roots of salt-stressed mung bean plants. In contrast to mung bean (and several other glycophytes), ornithine and arginine decarboxylase activity in roots of halophytes increased when plants were exposed to tap water or grown in a pre-washed soil—i.e. a hypotonic stress with respect to their natural habitat. NaCl, when present in the enzymatic assay mixture, inhibited arginine and ornithine decarboxylase in curde extracts of mung bean roots, but did not affect the activity of enzymes extracted from roots of the halophyte Pulicaria. Although no distinct separation between NaCl stress and osmotic stress could be made in the present study, the data suggest that changes in polyamines in response to NaCl stress in mung bean plants are coordinated at the organ level: activation of biosynthetic enzymes concomitant with increased putrescine biosynthesis from its precursors in the root system, and accumulation of putrescine in leaves of salt-stressed plants. In addition, hypertonic stress applied to glycophytes and hypotonic stress applied to halophytes both resulted in an increase in the activity of polyamine biosynthetic enzymes in roots.     

Comparison of the biosynthetic and biodegradative ornithine decarboxylases of Escherichia coli.

Biosynthetic ornithine decarboxylase was purified 4300-fold from Escherichia coli to a purity of approximately 85% as judged by polyacrylamide gel electrophoresis. The enzyme showed hyperbolic kinetics with a Km of 5.6 mM for ornithine and 1.0 micronM for pyridoxal phosphate and it was competitively inhibited by putrescine and spermidine. The biosynthetic decarboxylase was compared with the biodegradative ornithine decarboxylase [Applebaum, D., et al. (1975), Biochemistry 14, 3675]. Both enzymes were dimers of 80 000-82 000 molecular weight and exhibited similar kinetic properties. However, they differed significantly in other respects. The pH optimum of the biosynthetic enzyme was 8.1, compared with 6.9 for the biodegradative. Both enzymes were activated by nucleotides, but with different specificity. Antibody to the purified biodegradative ornithine decarboxylase did not cross-react with the biosynthetic enzyme. The evolutionary relationship of these two decarboxylases to the other amino acid decarboxylases of E. coli is discussed.     

Pseudomonas aeruginosa diaminopimelate decarboxylase: evolutionary relationship with other amino acid decarboxylases

The lysA gene encodes meso-diaminopimelate (DAP) decarboxylase (E.C.4.1.1.20), the last enzyme of the lysine biosynthetic pathway in bacteria. We have determined the nucleotide sequence of the lysA gene from Pseudomonas aeruginosa. Comparison of the deduced amino acid sequence of the lysA gene product revealed extensive similarity with the sequences of the functionally equivalent enzymes from Escherichia coli and Corynebacterium glutamicum. Even though both P. aeruginosa and E. coli are Gram-negative bacteria, sequence comparisons indicate a greater similarity between enzymes of P. aeruginosa and the Gram- positive bacterium C. glutamicum than between those of P. aeruginosa and E. coli enzymes. Comparison of DAP decarboxylase with protein sequences present in data bases revealed that bacterial DAP decarboxylases are homologous to mouse (Mus musculus) ornithine decarboxylase (E.C.4.1.1.17), the key enzyme in polyamine biosynthesis in mammals. On the other hand, no similarity was detected between DAP decarboxylases and other bacterial amino acid decarboxylases.      

Brain Polyamine Stress Response: Recurrence After Repetitive Stressor and Inhibition by Lithium

Abstract: We recently demonstrated that, unlike in peripheral tissues, the increase in activity of polyamine synthesizing enzymes observed in the brain after acute stress can be prevented by long-term, but not by short-term, treatment with lithium. In the present study we sought to examine the effects of chronic intermittent stress on two key polyamine synthesizing enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, and their modulation by lithium treatment. Adult male rats were subjected to 2 h of restraint stress once daily for 5 days and to an additional delayed stress episode 7 days later. Enzyme activities were assayed 6 h after the beginning of each stress episode. In contrast to the liver, where ornithine decarboxylase activity was increased (300% of the control) only after the first stress episode, the enzyme activity in the brain was increased after each stress episode (to ～170% of the control). Unlike ornithine decarboxylase activity, S-adenosylmethionine decarboxylase activity was slightly reduced after the first episode (86% of the control) but remained unchanged thereafter. After cessation of the intermittent stress period, an additional stress episode 7 days later led again to an increase in ornithine decarboxylase activity in the brain (225% of the control) but not in the liver, whereas S-adenosylmethionine decarboxylase activity remained unchanged. The latter increase in ornithine decarboxylase activity was blocked by lithium treatment during the intervening 7-day interval between stressors. The results warrant the following conclusions: (a) Repetitive application of stressors results in a recurrent increase in ornithine decarboxylase activity in the brain but to habituation of this response in the liver. (b) This brain polyamine stress response can be blocked by long-term (days) lithium treatment. (c) The study implicates an overreactive polyamine response as a component of the adaptive, or maladaptive, brain response to stressful events and as a novel molecular target for lithium action.     

Polyamine metabolism in Ancylostoma ceylanicum and Nippostrongylus brasiliensis

Spermidine was detected as the major polyamine of Ancylostoma ceylanicum as well as Nippostrongylus brasiliensis. Spermine was present in lower amounts whereas the level of putrescine was even less. S-Adenosylmethionine decarboxylase, a rate-limiting enzyme in the biosynthetic pathway of polyamines, was demonstrated at low levels in both parasites. Decarboxylation of lysine and arginine was absent or negligible and that of ornithine questionable, as the enzyme activity was not inhibited by alpha-difluoromethylornithine while RMI 71,645, an irreversible inhibitor of ornithine aminotransferase, strongly inhibited the liberation of CO2 from ornithine. High activity of ornithine aminotransferase was observed in both the parasites and may interfere with the assay for ornithine decarboxylase. Adults of A. ceylanicum were found to rapidly take up spermidine and spermine from incubation medium while uptake of putrescine was very low. These results indicate that hookworms depend on uptake and interconversion rather than de novo synthesis for their polyamine requirement.     

The enzymatic activities of the Escherichia coli basic aliphatic amino acid decarboxylases exhibit a pH zone of inhibition

The stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a "zone of inhibition" between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH.     

Stimulation by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine of rat hepatic polyamine biosynthesis in vivo

Intraperitoneal injection of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), resulted in a rapid and transient induction of rat hepatic ornithine decarboxylase (ODC) activity. Maximal activity was found about 5 hr after application. The levels of putrescine and spermidine increased accordingly, reaching a maximum at 7 and 12 hr following injection, respectively, while the concentration of spermine remained almost constant. The implications of these findings are discussed in relation to the mechanism of induction of ornithine decarboxylase and concomitant polyamine biosynthesis.     

Genetic approaches to the cellular functions of polyamines in mammals.

The polyamines putrescine, spermidine and spermine are organic cations shown to participate in a bewildering number of cellular reactions, yet their exact functions in intermediary metabolism and specific interactions with cellular components remain largely elusive. Pharmacological interventions have demonstrated convincingly that a steady supply of these compounds is a prerequisite for cell proliferation to occur. The last decade has witnessed the appearance of a substantial number of studies, in which genetic engineering of polyamine metabolism in transgenic rodents has been employed to unravel their cellular functions. Transgenic activation of polyamine biosynthesis through an overexpression of their biosynthetic enzymes has assigned specific roles for these compounds in spermatogenesis, skin physiology, promotion of tumorigenesis and organ hypertrophy as well as neuronal protection. Transgenic activation of polyamine catabolism not only profoundly disturbs polyamine homeostasis in most tissues, but also creates a complex phenotype affecting skin, female fertility, fat depots, pancreatic integrity and regenerative growth. Transgenic expression of ornithine decarboxylase antizyme has suggested that this unique protein may act as a general tumor suppressor. Homozygous deficiency of the key biosynthetic enzymes of the polyamines, ornithine and S-adenosylmethionine decarboxylase, as achieved through targeted disruption of their genes, is not compatible with murine embryogenesis. Finally, the first reports of human diseases apparently caused by mutations or rearrangements of the genes involved in polyamine metabolism have appeared.     

Activities and properties of putrescine-biosynthetic enzymes in Vibrio parahaemolyticus

The biosynthetic pathways for putrescine (Put) in Vibrio parahaemolyticus were delineated by measuring activities of the enzymes which would be involved in its biosynthesis. Experiments with labeled arginine and ornithine revealed that both of these amino acids were converted into Put by intact cells. The activities of three enzymes, arginine decarboxylase (ADC), ornithine decarboxylase (ODC), and agmatine ureohydrolase (AUH), were detected in cell extracts. ADC and ODC of V. parahaemolyticus were similar in the following properties to the corresponding enzymes of Escherichia coli: 1) both decarboxylases showed a pH optimum at 8.25 and required pyridoxal phosphate and dithiothreitol for full activity; 2) while ODC was considerably activated by GTP, ADC was only slightly; 3) both decarboxylases were inhibited by polyamines; 4) ADC was inhibited by difluoromethylarginine, a potent inhibitor of bacterial ADC. However, in contrast to the corresponding enzymes of E. coli, the V. parahaemolyticus ADC showed no requirement for Mg2+, and the AUH was active over a wide pH range of 8.5-9.5 with a maximum at pH 9.0. Furthermore, in all 6 strains tested, the activity of ADC was obviously high compared with that of ODC, and AUH was present with a relatively high activity. Cultivation of these strains at a suboptimal NaCl concentration (0.5%) resulted in a pronounced increase in both ADC and AUH activities. These observations suggest that the important pathway for Put biosynthesis in V. parahaemolyticus is the decarboxylation of arginine by ADC and the subsequent hydrolysis of its product, agmatine, by AUH.     

Polyamine Biosynthetic Enzymes in the Cell Cycle of Chlorella: Correlation between Ornithine Decarboxylase and DNA Synthesis at Different Light Intensities

During the life cycle of Chlorella vulgaris Beijerinck var vulgaris fa. vulgaris growing synchronously, the specific activity of ornithine decarboxylase peaked at the 2nd hour of the cycle, whereas that of arginine decarboxylase changed only slightly, increasing towards the end of the cycle. The endogenous level of putrescine and spermidine on a per cell basis increased gradually up to the 8th hour of the cycle, and declined thereafter. Thus, the peak of ornithine decarboxylase activity and the polyamine increase preceded both DNA replication (which took place between the 6th and 8th hours of the cycle) and autospore release (which started at the 8th hour). A 2-fold increase in the light intensity caused doubling of the DNA content, resulting in doubling of the number of autospores per mother cell. It also brought about a 2-fold increase in the specific activity of ornithine decarboxylase and polyamine content, the peaks being at the same hour of the cycle under high and low light intensities. The increase in cell number and polyamine content in a Chlorella culture grown under high light intensity was inhibited by α-difluoromethyl ornithine, a specific inhibitor of ornithine decarboxylase, this inhibition being partially reversed by putrescine.

It is suggested that in C. vulgaris the sequence of events which relates polyamine biosynthesis to cell division is as follows: increased ornithine decarboxylase activity, accumulation of polyamines, DNA replication, and autospore release.

     

Polyamine synthesis in maize cell lines

Uptake of [¹⁴C]putrescine, [¹⁴C]arginine, and [¹⁴C]ornithine was measured in five separate callus cell lines of Zea mays. Each precursor was rapidly taken into the intracellular pool in each culture where, on the average, 25 to 50% of the total putrescine was found in a conjugated form, detected after acid hydrolysis. Half-maximal labeling of each culture was achieved in less than 1 minute. Within this time frame of precursor incorporation, only putrescine derived from arginine was conjugated, indicating that putrescine pools derived from arginine may initially be sequestered from ornithine-derived putrescine. The decarboxylase activities were measured in each culture after addition of exogenous polyamine to the growth medium to assess differential regulation of the decarboxylases. Arginine and ornithine decarboxylase activities were augmented by added polyamine, the effect on arginine decarboxylase being eightfold greater than on ornithine decarboxylase. Levels of extractable ornithine decarboxylase were consistently 15- to 100-fold higher than arginine decarboxylase, depending on the titer of extracellular polyamine. Taken as whole the results support the idea that there are distinct populations of polyamine that are initially sequestered after the decarboxylase reactions and that give rise to separate end products and possibly have separate functions.