Escherichia coli mutants completely deficient in adenosylmethionine decarboxylase and in spermidine biosynthesis.

Mutants of Escherichia coli deficient in adenosylmethionine decarboxylase, an enzyme in the biosynthetic pathway for spermidine, were isolated after mutagenesis of E. coli K 12 with N-methyl-N-nitro-N-nitrosoguanidine or with the bacteriophage Mu. The mutated gene, designated speD, is at 2.7 min on the E. coli chromosome map. In several of the mutants resulting from Mu insertion both adenosylmethionine decarboxylase activity and spermidine were undetectable. The absence of spermidine from speD strains proves the essential role of adenosylmethionine decarboxylase in the biosynthetic pathway for spermidine. Despite the complete absence of spermidine, these mutants grew at 75% of the wild type rate.     

Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity

Unlike other eukaryotes, which can synthesize polyamines only from ornithine, plants possess an additional pathway from arginine. Occasionally non-enzymatic decarboxylation of ornithine could be detected in Arabidopsis extracts; however, we could not detect ornithine decarboxylase (ODC; EC 4. 1.1.17) enzymatic activity or any activity inhibitory to the ODC assay. There are no intact or degraded ODC sequences in the Arabidopsis genome and no ODC expressed sequence tags. Arabidopsis is therefore the only plant and one of only two eukaryotic organisms (the other being the protozoan Trypanosoma cruzi) that have been demonstrated to lack ODC activity. As ODC is a key enzyme in polyamine biosynthesis, Arabidopsis is reliant on the additional arginine decarboxylase (ADC; EC 4.1.1.9) pathway, found only in plants and some bacteria, to synthesize putrescine. By using site-directed mutants of the Arabidopsis ADC1 and heterologous expression in yeast, we show that ADC, like ODC, is a head-to-tail homodimer with two active sites acting in trans across the interface of the dimer. Amino acids K136 and C524 of Arabidopsis ADC1 are essential for activity and participate in separate active sites. Maximal activity of Arabidopsis ADC1 in yeast requires the presence of general protease genes, and it is likely that dimer formation precedes proteolytic processing of the ADC pre-protein monomer.     

1-Aminooxy-3-aminopropane reversibly prevents the proliferation of cultured baby hamster kidney cells by interfering with polyamine synthesis

The effects on cultured baby hamster kidney cells of 1-aminooxy-3-aminopropane, a potent new inhibitor of mammalian ornithine and S-adenosylmethionine decarboxylases and of spermidine synthase, were studied. At 0.5 mM concentration in the culture medium, the drug did not interfere with the transmethylation-transsulfuration pathway nor with the polyamine transport system, but it blocked the proliferation and macromolecule synthesis of the cells and reduced the cellular spermidine level to less than 10% of the control value at identical cell density. These changes were accompanied by a total cessation of the excretion of putrescine, spermidine, and acetylated polyamines into the culture medium, greatly increased activity of ornithine and S-adenosylmethionine decarboxylases, and an accumulation of both decarboxylated and intact S-adenosylmethionine. These effects were reversed by the removal of the inhibitor from the culture medium or by supplementing the medium with either 0.5 mM putrescine or 0.1 mM spermidine. In the former case, however, a lag period of 24 h was necessary for the cells to recover. The elevated concentration of decarboxylated S-adenosylmethionine normalized very slowly but apparently had no harmful effects on the cells. The clonigenic potential of the cells was only slightly reduced by prolonged treatment with 0.5 mM 1-aminooxy-3-aminopropane. Thus, the new drug is not toxic to the cells, but either directly or indirectly stops their proliferation by preventing the adequate formation of putrescine and spermidine.     

DL-a-Monofluoromethylputrescine is a potent irreversible inhibitor of Escherichia coli ornithine decarboxylase.

DL-alpha-Monofluoromethylputrescine (compound R.M.I. 71864) is an enzyme-activated irreversible inhibitor of the biosynthetic enzyme ornithine decarboxylase from Escherichia coli. This compound, however, has much less effect in vitro on ornithine decarboxylase obtained from Pseudomonas aeruginosa. These findings are in contrast with those previously found with the substrate analogue DL-alpha-difluoromethylornithine (compound R.M.I. 71782). The K1 of the DL-alpha-monofluoromethylputrescine for the E. coli ornithine decarboxylase is 110 microM, and the half-life (t1/2) calculated for an infinite concentration of inhibitor is 2.1 min. When DL-alpha-monofluoromethylputrescine is used in combination with DL-alpha-difluoromethylarginine (R.M.I. 71897), an irreversible inhibitor of arginine decarboxylase, in vivo in E. coli, both decarboxylase activities are inhibited (greater than 95%) but putrescine levels are only decreased to about one-third of control values and spermidine levels are slightly increased.     

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.     

Okadaic acid inhibits insulin stimulation of both ornithine decarboxylase and spermidine transport in hepatocyte cultures

• 1.1. Okadaic acid inhibited basal ODC activity in rat hepatocytes in culture and prevented any increase in ODC activity and in the rate of spermidine uptake promoted by both insulin and hypotonicity.
• 2.2. The increase promoted by AIB was not counteracted by okadaic acid.

     

In vitro effects of the polyamine biosynthesis inhibitor, α-difluoromethylornithine, on ornithine decarboxylase activities from three plant pathogenic fungi

The effects of α-difluoromethylornithine (DFMO) on in vitro ornithine decarboxylase (ODC) activities from three plant pathogenic fungi, Pyrenophora avenae, Pyricularia oryzae and Uromyces viciae-fabae , were studied. DFMO concentrations from 0·01 to 1·0 mmol/l produced no significant effects on ODC activities from the three fungi. However, increasing the DFMO concentration to 5 mmol/l produced a substantial reduction in in vitro ODC activity from Pyre, avenae. The ODC inhibitor, α-monofluoromethylornithine (2 mmol/l), significantly reduced in vitro ODC activity from Pyre. avenae , whereas RR-methyl acetylenic putrescine, an ODC inhibitor based on putrescine, produced no significant effect on the fungal enzyme.     

Inhibition of adenosylmethionine decarboxylase and perturbation of polyamine metabolism by 3-deaza-(+/-)aristeromycin

3-Deaza-(+/-)aristeromycin, previously known mainly as a potent inhibitor of adenosylhomocysteine hydrolase, can also inhibit the activity of adenosylmethionine decarboxylase. The release of [14C]CO2 from HeLa cells labeled with [carboxyl-14C]methionine was inhibited by more than 70% after 4 hours in the presence of 4 microM 3-deaza-(+/-)aristeromycin. Concomitant with this inhibition, there was a significant increase in the amount of putrescine in the HeLa cells. Adenosylmethionine decarboxylase isolated from HeLa cells could also be inhibited by 3-deaza-(+/-)aristeromycin and 3-deazaadenosine, 3-deazaadenosylhomocysteine, and 3-deaza-(+/-)aristeromycinylhomocysteine.     

Increased ornithine decarboxylase activity and polyamine biosynthesis are required for optimal cytolytic T lymphocyte induction

The objective of the present investigation was to evaluate the requirement for increased ornithine decarboxylase (ODC) activity and polyamine biosynthesis in the induction of cytolytic T lymphocytes (CTL). In this regard, we have utilized alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. DFMO treatment completely abrogated Con A-induced NW T-cell ODC activity. Similarly, DFMO treatment reduced putrescine and spermidine biosynthesis 100 and 87% respectively by the end of a 48-hr incubation period. Polyamine depletion reduced the Con A-mediated polyclonal induction of CTL by 52 and 81% at 24 and 48 hr of culture, respectively. The effect of DFMO on CTL induction could be reversed by the addition of exogenous putrescine. These data indicate that the observed effects of DFMO on CTL induction were mediated through inhibition of polyamine biosynthesis. Therefore, increased ODC activity and polyamine biosynthesis are required for optimal CTL induction. Furthermore, polyamine depletion did not impair IL-2 production; however, IL-2-dependent proliferation was reduced. These data are the first to discriminate between the requirement for polyamines with regard to IL-2 responsiveness, rather than IL-2 production, during a primary T-cell mitogenic response.     

Plasmodium falciparum: purification, properties, and immunochemical study of ornithine decarboxylase, the key enzyme in polyamine biosynthesis

Ornithine decarboxylase, the rate-limiting enzyme in the polyamine biosynthetic pathway has been purified 7,600 fold from Plasmodium falciparum by affinity chromatography on a pyridoxamine phosphate column. The partially purified enzyme was specifically tagged with radioactive DL-alpha-difluoromethylornithine and subjected to polyacrylamide gel electrophoresis under denaturing conditions. A major protein band of 49 kilodalton was obtained while with the purified mouse enzyme, a typical 53 kilodalton band, was observed. The catalytic activity of parasite enzyme was dependent on pyridoxal 5'-phosphate and was optimal at pH 8.0. The apparent Michaelis constant for L-ornithine was 52 microM. DL-alpha-difluoromethylornithine efficiently and irreversibly inhibited ornithine decarboxylase activity from P. falciparum grown in vitro or Plasmodium berghei grown in vivo. The Ki of the human malarial enzyme for this inhibitor was 16 microM. Ornithine decarboxylase activity in P. falciparum cultures was rapidly lost upon exposure to the direct product, putrescine. Despite the profound inhibition of protein synthesis with cycloheximide in vitro, parasite enzyme activity was only slightly reduced by 75 min of treatment, suggesting a relatively long half-life for the malarial enzyme. Ornithine decarboxylase activity from P. falciparum and P. berghei was not eliminated by antiserum prepared against purified mouse enzyme. Furthermore, RNA or DNA extracted from P. falciparum failed to hybridize to a mouse ornithine decarboxylase cDNA probe. These results suggest that ODC from P. falciparum bears some structural differences as compared to the mammalian enzyme.     

Hyperthermia and polyamine biosynthesis: decreased ornithine decarboxylase induction in skin and kidney after heat shock

The effect of hyperthermia treatments on ornithine decarboxylase (ODC) induction in mouse tissue was determined both in vitro and in vivo. In vitro, the addition of 12-0-tetradecanoylphorbol-13-acetate (TPA) to adult mouse skin pieces incubated at 37 degrees C in serum-free MEM led to a dramatic increase in epidermal ODC activity 5 hours following treatment. In contrast, incubation temperatures of 40 degrees C for the entire 5 hour incubation period rendered the skin pieces unresponsive to TPA for ODC induction. This inhibition of ODC induction was not the result of thermal skin kill, inactivation of TPA, or a general effect on epidermal protein synthesis. The inhibition of ODC induction could be reversed by switching the incubation temperature back to 37 degrees C. In vivo, raising the core body temperature in male mice to 41 degrees C for 1 hour resulted in a 78% decrease in kidney ODC activity. The kidney DNA synthesis and protein synthesis remained unaltered following the whole body hyperthermia treatments.     

Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.     

Cloning and characterization of spermidine synthase and its implication in polyamine biosynthesis in Helicobacter pylori strain 26695

The HP0832 (speE) gene of Helicobacter pylori strain 26695 codes for a putative spermidine synthase, which belongs to the polyamine biosynthetic pathway. Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), which serves as an aminopropyl donor. The deduced amino acid sequence of the HP0832 gene shares less than 20% sequence identity with most spermidine synthases from mammalian cells, plants and other bacteria. In this study, the HP0832 open reading frame (786 bp) was cloned into the pQE30 vector and overexpressed in Escherichia coli strain SG13009. The resulting N-terminally 6xHis-tagged HP0832 protein (31.9 kDa) was purified by Ni-NTA affinity chromatography at a yield of 15 mg/L of bacteria culture. Spermidine synthase activity of the recombinant protein was confirmed by the appearance of spermidine after incubating the enzyme with putrescine and dcSAM. Substrate specificity studies have shown that spermidine could not replace putrescine as the aminopropyl acceptor. Endogenous spermidine synthase of H. pylori was detected with an antiserum raised against the recombinant HP0832 protein. H. pylori strain 26695 contains putrescine and spermidine at a molar ratio of 1:3, but no detectable spermine or norspermidine was observed, suggesting that the spermidine biosynthetic pathway may provide the main polyamines in H. pylori strain 26695.     

A biochemical investigation of the adenovirus-induced G1 to S phase progression: Thymidine kinase,ornithine decarboxylase,and inhibitors of polyamine biosynthesis

Biochemical events were investigated in the G1 to S phase progression induced in quiescent rodent cells by human adenovirus type 5 (Ad5) and by serum. Thymidine kinase activity increased after infection of cells with Ad5 or addition of 10% serum. These stimulations were additive. An early viral gene was respnsible for induction by Ad5, but the early mutants ts36, ts37, and ts125 induced thymidine kinase at the permissive and nonpermissive temperatures. Several differences were found between cells stimulated by serum compared with Ad5. Induction of thymidine kinase was delayed in Ad5- infected cells, insensitive to 0.01 μ/ml actinomycin D and relatively resistant to reduced Ca²⁺ compared with induction by serum. Ornithine decarboxylase was induced by serum, but not by Ad5. α-Methylornithine had little effect on the induction of thymidine kinase by Ad5, but reduced the induction of thymidine kinase by serum, suggesting that Ad5-induced entry into S phase is uncoupled from polyamine biosynthesis. Methylglyoxal bis(guanylhydrazone), however, prevented the induction of thymidine kinase by both serum and Ad5. Adenovirus infection appears to induce cellular DNA synthesis and thymidine kinase in G1-arrested cells by a mechanism different from serum, and by passes events in the normal G1 to S phase progression.     

Inhibition of polyamine biosynthesis in Acanthamoeba castellanii and 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by chitosanoligosaccharide

Growth of Acanthamoeba castellaniiwas inhibited by chitosanoligosaccharide (up to 20 mg ml^–1) from the shells of crabs but was reversed by the polyamines, putrescine or spermidine, at 0.8 mM. Chitosanoligosaccharide strongly inhibited the induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate, a key enzyme of polyamine biosynthesis, which is enhanced in tumour promotion.     

The effects of 1-aminooxy-3-aminopropane and S-(5'-deoxy-5'-adenosyl)methylthioethylhydroxylamine on ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase from Escherichia coli

1-Aminooxy-3-aminopropane (APA) was shown to be a potent competitive inhibitor (Ki = 1.0 nM) of partially purified Escherichia coli ornithine decarboxylase. APA did not inhibit S-adenosyl-L-methionine decarboxylase and spermidine from E. coli. S-(5'-Deoxy-5'-adenosyl)methylthioethylhydroxylamine (AMA), which is a structural analogue of decarboxylated S-adenosyl-L-methionine, was for the first time shown to be an irreversible inhibitor of bacterial S-adenosyl-L-methionine decarboxylase and a competitive inhibitor (Ki = 47 microM) of bacterial ornithine decarboxylase. AMA had no effect on spermidine synthase.     

Effect of alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on polyamine levels in rat tissues.

α-Difluoromethylornithine (RMI 71.782), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (E.C. 4.1.1.17) $\text{in vitro}$, causes a rapid, long-lasting, dose-dependent decrease of ornithine decarboxylase activity in prostate and, to a lesser extent, in thymus and testis of rats when injected intraperitoneally. Repeated doses (100 mg/kg or 1 g/kg) of α-difluoromethylornithine given to rats for two weeks markedly decreased polyamine concentrations in several rat tissues and selectively slowed down growth of ventral prostate and of thymus.     

Polyamines in brain and heart of the neonatal rat: effects of inhibitors of ornithine decarboxylase and spermidine synthase

Daily administration of dicyclohexylamine (DCHA), an inhibitor of spermidine synthase, to neonatal rats produced a dose-dependent depletion of brain spermidine, accompanied by a rise in putrescine and spermine. Despite continued DCHA treatment, levels of all three polyamines returned toward normal within two weeks. alpha-Difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, had a much more profound and persistent effect on spermidine and also depleted putrescine throughout drug administration; furthermore, DFMO prevented both the elevation of putrescine caused by DCHA and the eventual restitution of spermidine levels. Although a similar pattern of effects was seen in the heart, the time course of onset of DCHA-induced alterations in polyamine levels and the rapidity of subsequent adaptation were considerably different from those in brain. The net activity of DCHA toward polyamines in developing tissues thus involves the direct actions of the drug on spermidine synthesis in combination with compensatory metabolic adjustments made by each tissue to polyamine depletion.     

Effects of dexamethasone on spermidine N1-acetyltransferase and ornithine decarboxylase activities in rat spleen

Treatment of rats with the glucocorticoid dexamethasone causes an increase in the activity of cytosolic spermidine N¹-acetyltransferase both in the spleen and thymus, but not, however, in liver, kidney or lung. The induced spermidine N¹-acetyltransferase activity in the spleen catalyses acetylation of spermidine as well as spermine and sym-norspermidine, but not of diamines and histones. The enzyme induction depends on the dose of dexamethasone, and is suppressed by cycloheximide, which suggests that de novo protein synthesis is required for the action of this glucocorticoid. N¹-acetylspermidine accumulates in the spleen after dexamethasone treatment, while spermidine progressively decreases and is partly converted into putrescine, the content of which transiently increases. In accordance with previous reports, dexamethasone was found to cause a rapid and large fall in the activity of spleen ornithine decarboxylase which was effected via the appearance of an inhibitor of the enzyme. Glucocorticoids exert large catabolic effects on lymphoid tissues, and further selectively affect the activities of spermidine N¹-acetyltransferase and ornithine decarboxylase in the thymus and spleen. These latter selective responses may represent an important early event in lymphoid tissue response to glucocorticoid hormones.     

The modulation of the induction of ornithine decarboxylase by spermine,spermidine and diamines

Extremely low concentrations of putrescine, spermidine and spermine added to the extracellular medium of cultures of mammalian cells inhibit the induction of ornithine decarboxylase activity despite 100- to 1,000-fold greater intracellular polyamine concentrations. The diamines, 1,2-diaminoethane, 1,3-diaminopropane, 1,5-diaminopentane, 1,7-diaminoheptane, 1,10-diaminodecane, 1,12-diaminododecane also inhibit ornithine decarboxylase at all concentrations tested (greater than 10^?6 M). In contrast, 10^?6 M to 10 ^?3 M 1,8-diaminooctane, the alkyl analog of spermidine, enhances ornithine decarboxylase activity. The concentraton of putrescine required to inhibit the activity of ornithine decarboxylase by 50% is a characteristic of each cell line; however, it varies by as much as 1,000-fold among the five cell lines we have tested (L1210 leukemic, H35 hepatoma, N18 neuroblastoma, W256 carcinosarcoma and 3T3 fibroblasts). The antizyme to ornithine decarboxylase can be induced in all these cells by high (di)(poly)amine concentrations. Based on these and other experiments we suggest a working hypothesis: that the polyamines regulate ornithine decarboxylase activity through two different sites that may be interrelated; a sensitive membrane-mediated site that responds to minute fluctuations of extracellular polyamine levels and a coarse site which may be intracellular or membrane associated that responds to larger fluctuations of intracellular polyamine levels. The consequences of such a control mechanism operating within the whole organism are discussed.