1-Aminooxy-3-aminopropane, a new and potent inhibitor of polyamine biosynthesis that inhibits ornithine decarboxylase, adenosylmethionine decarboxylase and spermidine synthase

1-Aminooxy-3-aminopropane was shown to be a potent competitive inhibitor (Ki = 3.2 nM) of homogenous mouse kidney ornithine decarboxylase, a potent irreversible inhibitor (Ki = 50 microM) of homogeneous liver adenosylmethionine decarboxylase and a potent competitive (Ki = 2.3 microM) of homogeneous bovine brain spermidine synthase. It did not inhibit homogeneous bovine brain spermine synthase and it did not serve as a substrate for spermidine synthase. The compound did not inhibit tyrosine aminotransferase, alanine aminotransferase or aspartate aminotransferase, which are pyridoxal phosphate-containing enzymes like ornithine decarboxylase. The inactivation of adenosylmethionine decarboxylase was partially prevented by pyruvate, which is the coenzyme of adenosylmethionine decarboxylase, and by the substrate, adenosylmethionine. 1-Aminooxy-3-aminopropane at 0.5 mM concentration inhibited the growth of HL-60 promyelocytic leukemia cells and this inhibition was prevented by spermidine but not by putrescine.     

Restriction of bacterial growth by inhibition of polyamine biosynthesis by using monofluoromethylornithine, difluoromethylarginine and dicyclohexylammonium sulphate.

Bacterial growth was measurably slowed by a combination of drugs which inhibit polyamine-biosynthetic enzymes. Addition of DL-alpha-monofluoromethylornithine, which was shown to inactivate irreversibly ornithine decarboxylase extracted from Escherichia coli (Ki = 0.36 mM) and Pseudomonas aeruginosa (Ki = 0.30 mM), DL-alpha-difluoromethylarginine and dicyclohexylammonium sulphate to cultures of E. coli or P. aeruginosa resulted in a 40 and 70% increase in generation times (decreased growth rates) respectively, which was completely reversed by the addition of 0.1 mM-putrescine plus 0.1 mM-spermidine to the medium. Decreased intracellular polyamine concentrations correlated with increased generation times; putrescine concentration was decreased by 70% in E. coli and 80% in P. aeruginosa, while spermidine concentration was decreased by 50% in E. coli and 95% in P. aeruginosa. Subsequent investigation of the inactivation of the ornithine decarboxylase by monofluoromethylornithine indicated that it was active-site directed, as the normal substrate ornithine slowed the rate of inhibition. Specific interference with polyamine biosynthesis may be a viable approach to control of some bacterial infections.     

Interaction of alkylputrescines with ornithine decarboxylase from rat liver and Escherichia coli: an in vitro and in vivo study

The inhibitory effect of a series of 2-alkylputrescines on rat liver and Escherichia coli ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined. At 2.5 mM concentrations, 2-methyl-, 2-propyl-, 2-butyl-, 2-pentyl- and 2-hexylputrescines were stronger inhibitors of the mammalian enzyme than putrescine. Only the higher homologues (from 2-propyl- to 2-hexylputrescine) were inhibitors of the E. coli enzyme. An analysis of the effect of increasing concentrations of the 2-alkylputrescines showed that the main difference in the behaviour of the mammalian and E. coli decarboxylases toward 2-alkylputrescines was that the former was strongly inhibited by 2-methylputrescine whereas the latter was not. 2-Alkylputrescines were found to be competitive inhibitors of both the bacterial and mammalian enzyme. The smallest Ki values (0.1 and 0.5 mM) were found for the 2-hexyl- and 2-pentylputresciens. N-Methyl-, N-ethyl-, N-propyl- and N-butylputrescines (50 mumol per 100 g body weight) were assayed as inhibitors of thioacetamide-induced rat liver ornithine decarboxylase. N-Propylputrescine was found to be the most inhibitory (66% inhibition) and although the N-alkylputrescines were taken up by the liver, they did not inhibit the liver polyamine pools. Both putrescine and N-methylputrescine were found to stabilize the thioacetamide-induced ornithine decarboxylase at the onset of the enzyme's degradation, while 2-alkylputrescines were inhibitory under similar conditions. N-Methylputrescine induced antizyme in thioacetamide-treated rats. In thioacetamide- or dexamethasone-treated rats, 2-methylputrescine was found to be the strongest in vivo inhibitor of the liver decarboxylase. Although 2-alkylputrescines were efficiently taken up by the liver, they did not noticeably inhibit its polyamine pools. 2-methylputrescine decreased the putrescine concentration of the liver, but not its spermidine and spermine content. No induction of ornithine decarboxylase antizyme by 2-methylputrescine could be detected. The intrahepatic concentration of the latter decreased with time, very likely due to its degradation by a diamine oxidase, since the decrease was inhibited by aminoguanidine.     

Effect of N-alkyl and C-alkylputrescines on the activity of ornithine decarboxylase from rat liver and E. coli

N-Methyl-, N-ethyl-, N-propyl-, N-butyl-, N,N-dimethyl- and N,N'-dimethylputrescines were assayed as inhibitors of ornithine decarboxylase (EC 4.1.1.17) from rat liver and from Escherichia coli. They were found to be poor inhibitors, with the exception of N-propylputrescine and N,N-dimethylputrescine, which were inhibitory at 25 mM. A homologous series of 1-alkylputrescines ranging from 1-methylputrescine (1,4-diaminopentane) to 1-heptylputrescine (1,4-diaminoundecane) was assayed for effect on the activity of ornithine decarboxylase from the same sources. 1-Methylputrescine (5 mM) inhibited the mammalian enzyme, while the higher homologues showed significantly less inhibitory activity. When assayed on the bacterial enzyme, 1-methylputrescine (5 mM) was not inhibitory, while the higher homologues showed inhibitory effects. At higher concentrations, 1-methylputrescine and 1-heptylputrescine were the best inhibitors of these series of rat liver ornithine decarboxylase. When 1-methylputrescine, 2-methylputrescine, 1,2-dimethylputrescine, 1,3-dimethylputrescine and 1,4-dimethylputrescine were assayed as inhibitors of the decarboxylase, 2-methylputrescine was found to be the best inhibitor of the rat liver enzyme, while 1,3-dimethylputrescine was the best inhibitor of the bacterial enzyme. 1,4-Dimethylputrescine (2,5-diaminohexane) did not inhibit the enzyme from either source. Both, 2-methylputrescine and 1-methylputrescine, as well as the 1,2- and 1,3-dimethylputrescines were competitive inhibitors of the enzyme, and a Ki of 1 mM was obtained for 2-methylputrescine when the rat liver decarboxylase was used. N-Methyl, 1-methyl and 2-methylputrescines were found to inhibit in vivo the activity of rat liver ornithine decarboxylase which had been previously induced by thioacetamide treatment. 2-Methylputrescine (50 mumol/100 g body weight) was found to be the best in vivo inhibitor (93% inhibition), while putrescine under similar conditions inhibited 56% of the enzymatic activity.     

Hormonal control of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase during development of the rat epididymis

The specific activities of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase were determined during growth of the rat epididymis. Ornithine decarboxylase activity was first detectable on day 21 and increased 10-fold in both the head and tail of epididymis prior to their rapid growth responses. Hypophysectomy reduced ornithine decarboxylase activity to undetectable levels, but enzyme activity was restored by treatment with gonadotropins or testosterone. Testosterone also induced a precocious 10-fold increase of epididymal ornithine decarboxylase in the pre-pubertal rat. In contrast, the specific activity of S-adenosyl-L-methionine decarboxylase changed little during development and merely doubled in response to hormonal treatments. The results describe a pattern of changes in these enzyme activities during hormone-dependent development of the epididymis, and suggest that ornithine decarboxylase is the rate-limiting activity in the regulation of spermidine biosynthesis by testosterone in this organ.     

Polyamines and their biosynthetic enzymes in Ehrlich ascites-carcinoma cells. Modification of tumour polyamine pattern by diamines.

1. Ehrlich ascites-carcinoma cells contained relatively high concentrations of spermidine and spermine, but the putrescine content of the washed cells was less than 10% of that of higher polyamines. 2. Ascites-tumour cells likewise exhibited high activities of L-ornithine decarboxylase (EC 4.1.1.17), S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50), spermidine synthase (EC 2.5.1.16) and spermine synthase. 3. During the first days after the inoculation, the polyamine pattern of the ascites cells was characterized by a high molar ratio of spermidine to spermine, which markedly decreased on aging of the cells. 4. Various diamines injected into mice bearing ascites cells rapidly and powerfully decreased ornithine decarboxylase activity in the carcinoma cells, apparently through a mechanism that was not a direct inhibition of the enzyme in vitro. Cadaverine (1,5-diaminopentane) and 1,6-diaminohexane were the most potent inhibitors of ornithine decarboxylase among the amines tested. 5. Chronic treatment of the mice with diamines resulted in a virtually complete disappearance of ornithine decarboxylase activity, and after 24h a significant decline in spermidine accumulation. 6. Cadaverine appeared to be an especially suitable compound for use as an inhibitor of the synthesis of higher polyamines, at least in Ehrlich ascites cells, since this diamine also acted as a competitive inhibitor for putrescine in the spermidine synthase reaction without being incorporated into the higher polyamines.     

GTP-insensitive ornithine decarboxylase in acetobacteria able to synthesize spermine

Several Acetobacteria contained large amounts of spermine in addition to the putrescine and spermidine, which are the polyamines normally found in prokaryotes. A spermine synthase present in cell extracts of these Acetobacteria is the first example of this enzyme in prokaryotes. Dicyclohexylammonium sulphate inhibited both spermidine synthase and spermine synthase activities in Acetobacteria. Their ornithine decarboxylase was not stimulated by GTP nor inhibited by ppGpp and pppGpp (magic spots I and II) in contrast to ornithine decarboxylase of nearly all bacteria studied so far. However, their S-adenosyl-L-methionine decarboxylase resembled other prokaryotic adenosylmethionine decarboxylases in requiring Mg2+ ions in vitro for full activity.     

Effect of DL-alpha-hydrazino-delta-aminovaleric acid, an inhibitor of ornithine decarboxylase, on polyamine metabolism in isoproterenol-stimulated mouse parotid glands.

The effects of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA) on polyamine metabolism in isoproterenol(IPR)-stimulated mouse parotid glands were investigated both in vitro and in vivo. Using partially enzyme preparations, it was found that DL-HAVA strongly inhibited ornithine decarboxylase (EC 4.1.1.17) by competing with L-ornithine. Other enzymes metabolizing ornithine and pyridoxal phosphate-dependent enzymes were at least 2-3 orders of magnitude less sensitive to DL-HAVA than ornithine decarboxylase. Administration of DL-HAVA greatly depressed the increases in both the putrescine level and putrescine formation from L-ornithine induced by IPR in the mouse parotid glands. Under the same conditions, the stimulation of DNA synthesis and subsequent cell proliferation in the glands were also suppressed. However, the IPR-dependent increases in S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activity, synthesis and the tissue concentration of spermidine, and RNA synthesis in the parotid glands were not affected appreciably by DL-HAVA. The inhibition of DNA synthesis by DL-HAVA was effectively prevented by putrescine, but not by spermidine or 1,7-diaminoheptane, given at the same time when DL-HAVA inhibited stimulation of putrescine formation by IPR. From these results, it is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine. The effects of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl-L-methionine decarboxylase, on the metabolism of polyamines and nucleic acids in growing parotid glands were also examined.     

Polyamine metabolism in MRC5 cells infected with different herpesviruses.

Both methyglyoxal bis(guanylhydrazone), an inhibitor of S-adenosyl-L-methionine decarboxylase (EC.4.1.1.50) and DL-α-methylornithine, an inhibitor of ornithine decarboxylase (EC.4.1.1.17), are shown to be potent inhibitors of the replication of human cytomegalovirus (HCMV) in MRC-5 cells. These compounds, both inhibitors of polyamine biosynthesis, do not affect the replication of either herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). This difference in antiviral effect is shown to be related to the stimulation of spermidine and spermine synthesis in host cells following HCMV infection and the inhibition of polyamine metabolism in HSV-1 or HSV-2-infected cells. Inhibition of HCMV replication by the inhibitors of polyamine biosynthesis is accompanied by a marked decrease in the formation of intranuclear, DNA-containing inclusions characteristic of HCMV infection. These results suggest significant differences in the mechanisms of replication of different herpesviruses.     

Cloning and sequence analysis of the meso-diaminopimelate decarboxylase gene from Bacillus methanolicus MGA3 and comparison to other decarboxylase genes.

The lysA gene of Bacillus methanolicus MGA3 was cloned by complementation of an auxotrophic Escherichia coli lysA22 mutant with a genomic library of B. methanolicus MGA3 chromosomal DNA. Subcloning localized the B. methanolicus MGA3 lysA gene into a 2.3-kb SmaI-SstI fragment. Sequence analysis of the 2.3-kb fragment indicated an open reading frame encoding a protein of 48,223 Da, which was similar to the meso-diaminopimelate (DAP) decarboxylase amino acid sequences of Bacillus subtilis (62%) and Corynebacterium glutamicum (40%). Amino acid sequence analysis indicated several regions of conservation among bacterial DAP decarboxylases, eukaryotic ornithine decarboxylases, and arginine decarboxylases, suggesting a common structural arrangement for positioning of substrate and the cofactor pyridoxal 5'-phosphate. The B. methanolicus MGA3 DAP decarboxylase was shown to be a dimer (M(r) 86,000) with a subunit molecular mass of approximately 50,000 Da. This decarboxylase is inhibited by lysine (Ki = 0.93 mM) with a Km of 0.8 mM for DAP. The inhibition pattern suggests that the activity of this enzyme in lysine-overproducing strains of B. methanolicus MGA3 may limit lysine synthesis.     

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 S-adenosyl-L-methionine decarboxylase by 1-aminooxy-3-aminopropane: enzyme kinetics and effects on the enzyme activity in cultured cells

The kinetics of inactivation of adenosylmethionine decarboxylase of rat liver and of baby hamster kidney cells (BHK21/C31) by 1-aminooxy-3-aminopropane was studied. The apparent dissociation constants (Ki) for the hepatic and BHK21/C13 enzymes were 1.5 and 2.0 mM and the times of half-inactivation at infinite concentration of the inhibitor (tau 1/2) were 1.2 and 3.8 min, respectively. Treatment of BHK21/C13 with 0.5 mM 1-aminooxy-3-aminopropane prevented cell growth and depleted the cells of putrescine and spermidine within 1 day. The depletion of spermidine resulted in increased activity of S-adenosylmethionine decarboxylase which was due, at least partly, to the increase in the half-life of the enzyme activity. Because spermine levels were not significantly affected, it appears that spermidine is the principal feedback regulator of S-adenosylmethionine decarboxylase. So, 1-aminooxy-3-aminopropane is a very weak inhibitor of S-adenosylmethionine decarboxylase and the cellular effects can be correlated primarily with its inhibitory effects on ornithine decarboxylase and spermidine synthase. In cell-free systems, however, 1-aminooxy-3-aminopropane is likely to find use in unraveling the reaction mechanism of S-adenosylmethionine decarboxylase.     

Feedback regulation of S-adenosylmethionine decarboxylase synthesis.

Treatment of Ehrlich ascites-tumour cells with 1-amino-oxy-3-aminopropane (AOAP), a potent inhibitor of ornithine decarboxylase, resulted in a marked decrease in cellular contents of putrescine and spermidine, concomitant with an arrest of cell growth. The activity of S-adenosylmethionine decarboxylase (AdoMetDC) was greatly increased in cells treated with AOAP. This increase in AdoMetDC activity was shown to be, at least partly, caused by enhanced synthesis of the enzyme, which most likely was induced by the change in cellular polyamine content.     

Trypanosoma brucei ornithine decarboxylase: enzyme purification, characterization, and expression in Escherichia coli

Ornithine decarboxylase from the African trypanosome is an important target for antitrypanosomal chemotherapy. Despite this, the enzyme had not been previously purified or extensively characterized as it is a very low level protein. In this paper we describe the purification of Trypanosoma brucei brucei ornithine decarboxylase from bloodstream form trypomastigotes by 107,000-fold to a specific activity of 2.7 x 10(6) nmol CO2/h/mg of protein in the parasite. T. brucei ornithine decarboxylase had a native molecular weight of 90,000 and a subunit molecular weight of 45,000. The isoelectric point of the protein was 5.0. The Km for ornithine was 280 microM and the Ki for the irreversible inhibitor alpha-difluoromethylornithine (DFMO) was 220 microM with a half-time of inactivation at saturating DFMO concentration of 2.7 min. T. brucei ornithine decarboxylase appears similar to mouse ornithine decarboxylase, further supporting our previous suggestion that the selective toxicity of DFMO to the parasite is not due to catalytic differences between the two proteins. Although a small quantity of T. brucei ornithine decarboxylase was purified from T. brucei, extensive structural and kinetic studies will require a more ample source of the enzyme. We therefore expressed our previously cloned T. brucei ornithine decarboxylase gene in Escherichia coli using a vector that contains an inducible lambda promoter. T. brucei ornithine decarboxylase activity was induced in E. coli to levels that were 50 to 200 fold of that present in the long-slender bloodstream form of T. brucei. Ornithine decarboxylase activity in the crude E. coli lysate was 1500-6000 nmol of CO2/h/mg of protein and represented 0.05-0.2% of the total cell protein. The recombinant T. brucei ornithine decarboxylase was purified to apparent homogeneity from the transformed E. coli. The purified recombinant enzyme had kinetic and physical properties essentially identical to those of the native enzyme.     

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.     

Ornithine decarboxylase from Saccharomyces cerevisiae. Purification, properties, and regulation of activity

Ornithine decarboxylase has been purified 1,500-fold to homogeneity from a spe2 mutant of Saccharomyces cerevisiae which lacks S-adenosylmethionine decarboxylase and is derepressed for ornithine decarboxylase. The ornithine decarboxylase is a single polypeptide (Mr = 68,000) and requires a thiol and pyridoxal phosphate for activity. Addition of 10(-4) M spermidine and 10(-4) M spermine to the growth medium reduces the activity of the enzyme by 90% in 4 h. However, immunoprecipitation studies showed that the extracts of polyamine-treated cells contain as much enzyme protein as normal cell extracts. This loss of ornithine decarboxylase activity is probably due to a post-translational modification of enzyme protein because we found no evidence for any inhibitor of activity in the polyamine-treated cells.     

Arginine decarboxylase from a Pseudomonas species.

An arginine decarboxylase has been isolated from a Pseudomonas species. The enzyme is constitutive and did not appear to be repressed by a variety of carbon sources. After an approximately 40-fold purification, the enzyme appeared more similar in its properties to the Escherichia coli biosynthetic arginine decarboxylase than to the E. coli inducible (biodegradative) enzyme. The Pseudomonas arginine decarboxylase exhibited a pH optimum of 8.1 and an absolute requirement of Mg2+ and pyridoxal phosphate, and was inhibited significantly at lower Mg2+ concentrations by the polyamines putrescine, spermidine, and cadaverine. The Km for L-arginine was about 0.25 mM at pH 8.1 AND 7.2. The enzyme was completely inhibited by p-chloromercuribenzoate. The inhibition was prevented by dithiothreitol, a feature that suggests the involvement of an -SH group. Of a variety of labeled amino acids tested, only L-arginine, but not D-arginine was decarboxylated. D-Arginine was a potent inhibitor of arginine decarboxylase with a Ki of 3.2 muM.     

Non-competitive inhibition of ornithine decarboxylase by a phosphopeptide and phosphoamino acids

In Tetrahymena pyriformis the cytosolic ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity is considerably inhibited by the presence of polyamines in the growth medium, while the nuclear ornithine decarboxylase is only slightly affected. Experimental evidence suggests that the presence of putrescine and/or spermidine elicits the appearance of non-competitive inhibitors of ornithine decarboxylase. One of the inhibitors has a molecular weight of 25,000 and properties of antizyme. In addition, two other low molecular weight inhibitors are extracted, one which is a phosphoserine oligopeptide, and the other which is phosphotyrosine. All inhibit non-competitively the homologous and heterologous (Escherichia coli and rat liver) ornithine decarboxylases. Similarly, non-competitive inhibition was obtained when the commercially available phosphoamino acids were tested against the already mentioned ornithine decarboxylases.