Interconversion Of Polyamines Research Articles

Polyamine metabolism as chemotherapeutic target in protozoan parasites.

Polyamines are essential cell constituents for all organisms. The present review highlights important differences in the synthesis, degradation, and interconversion of polyamines between the protozoan parasites (Trypanosoma brucei, Trypanosoma cruzi, Cryptosporidium parvum and Trichomonas vaginalis) and their mammalian hosts. Approaches include development of mono- and di-substituted polyamine analogs targeting polyamine interconversion, as well as more traditional targeting of synthetic enzymes and related pathways.

Agmatine modulates the in vivo biosynthesis and interconversion of polyamines and cell proliferation

Agmatine has recently gained wide interest as a bioactive arginine metabolite with a multitude of physiological functions. This study evaluates the in vivo role of agmatine in the modulation of metabolism and intracellular level of polyamines . Here, we report that agmatine, administered to mice, differentially affects the renal and liver activity of the two key enzymes regulating polyamine biosynthesis and interconversion/degradation. Thus, agmatine exerts a negative regulation of ODC activity and protein content, and positive regulation of SSAT activity, having no effect on ODC and SSAT transcript level. Agmatine modulation of ODC and SSAT activities is noticeably augmented by the inhibitor of its catabolism, aminoguanidine. Antizyme and eIF4E protein content appears to be affected by agmatine only insignificantly and apparently do not contribute to agmatine-induced down-regulation of ODC content. The homeostasis of spermidine and spermine is preserved after agmatine injection, while the putrescine level decreases. Furthermore, when tested in a mouse kidney injury model, agmatine, partially but significantly, reduces [ 3H] thymidine incorporation into DNA. This is consistent with suppressed renal tubule epithelial cell proliferation. The findings provide in vivo evidence of a substantial role of agmatine as a modulator of polyamine biosynthesis and degradation and suggest its suppressive effect on cell proliferation.

Arginase expression in peritoneal macrophages and increase in circulating polyamine levels in mice infected with Schistosoma mansoni.

We investigated the nitric oxide (NO) synthase and arginase pathways in resident peritoneal macrophages of mice infected with the tropical parasite Schistosoma mansoni. The two enzymes may have opposite effects, insofar as NO may be involved in the killing of the parasite whereas arginase may stimulate parasite growth via polyamine synthesis. We determined the effects of the infection on the expression and activity of the two enzymes in macrophages, before and after cytokine activation. Cells from infected mice expressed the hepatic type I arginase, whereas in control cells, the enzyme was expressed only after cytokine activation, as were NO synthase II and type II arginase in both groups of cells. Moreover, we found that in infected mice, arginase expression in macrophages was associated with a ten fold increase in the concentration of circulating ornithine-derived polyamines. This may be of pathological importance, since parasitic helminths are though to be dependent on their hosts for the uptake and interconversion of polyamines.

Aging and polyamine acetylation in rat kidney

The acetylation of polyamines was investigated in rat kidney as a function of age. The activity of cytosolic spermidine/spermine N 1-acetyltransferase, the rate-limiting enzyme in polyamine interconversion, increased from 3 to 36 months of age. The activity of cytosolic spermidine N 8-acetyltransferase, an enzyme probably related to polyamine excretion, also increased. The activity of polyamine oxidase, which catalyzes the oxidative cleavage of polyamine N'-acetyl derivatives into putrescine, decreased until 24 months, when an accumulation of N 1-acetylspermidine occurred. Subsequently, at 36 months, polyamine oxidase activity returned toward high values, in concomitance with the disappearance of N 1-acetylspermidine, an increase in spermidine and putrescine, and a decline in spermine was observed. Our results show that in rat kidney during aging there is an activation of the acetylation and interconversion of higher polyamines into putrescine, which is considered an alternative pathway of spermidine and putrescine formation.

Synthesis and evaluation of unsymmetrically substituted polyamine analogues as modulators of human spermidine/spermine-N1-acetyltransferase (SSAT) and as potential antitumor agents.

Spermidine/spermine-N1-acetyltransferase (SSAT), the rate-limiting step in polyamine catabolism, is critical for the interconversion and modulation of cellular polyamines. Inhibitor-initiated induction of this enzyme also appears to correlate with the sensitivity of tumor cells to a class of novel polyamine analogues, the bis(ethyl)polyamines. Thus, terminally alkylated polyamines which modulate the cellular level of SSAT could be of great value for understanding the role of this enzyme both in analogue-mediated cytotoxicity and in overall cellular polyamine metabolism. Such analogues could also become important therapeutic agents by disrupting cellular polyamine metabolism. The structure-activity relationships defining the interaction of polyamine analogues with SSAT have not been fully elucidated, and, in particular, unsymmetrically alkylated polyamines have not been synthesized and evaluated as modulators of SSAT. To this end, we now report the synthesis and preliminary biological evaluation of N1-ethyl-N11-propargyl-4,8-diazaundecane and N1-ethyl-N11-((cyclopropyl)methyl)-4,8-diazaundecane via a synthetic pathway which represents an efficient route to a variety of unsymmetrically substituted polyamine analogues. The title compounds act as effective inhibitors of isolated human SSAT and produce a differential superinduction of SSAT in situ which appears to be associated with a cell specific cytotoxic response in two human lung cancer cell lines. In so doing, these analogues exhibit promising antitumor activity against cultured human lung cancer cells.

Purification and characterization of polyamine oxidase from Ascaris suum.

The interconversion of polyamines in the parasite nematode Ascaris suum by a novel type of polyamine oxidase was demonstrated. The nematode enzyme was clearly distinguishable from monoamine and diamine oxidases as well as from the mammalian polyamine oxidase, as shown by the use of the specific inhibitors pargyline, aminoguanidine and MDL 72527 respectively. All three inhibitors had no effect on the parasite polyamine oxidase, and the enzyme did not accept diamines such as putrescine, cadaverine or histamine as substrates. The parasite polyamine oxidase selectively oxidizes spermine and spermidine but not N-acetylated polyamines, whereas the mammalian tissue-type polyamine oxidase shows preference for the N-acetylated polyamines. These results suggest a regulatory function of the nematode polyamine oxidase in the degradation and interconversion of polyamines in parasite nematodes. The enzyme was purified to homogeneity by gel filtration, preparative isoelectric focusing and subsequent affinity chromatography on spermine- and berenil-Sepharose 4B. With respect to reaction type, the prosthetic group FAD, the molecular mass (66 kDa) and the contents of thiol and carbonyl groups, the polyamine oxidase from A. suum is similar to the isofunctional enzyme of mammalian tissue.

Protection from chlordecone-amplified carbon tetrachloride toxicity by cyanidanol: Regeneration studies

Previous work has shown that chlordecone (CD)-amplified CCl 4 hepatotoxicity and lethality can be mitigated by pretreatment with cyanidanol. These studies also revealed that stimulated hepatocellular regeneration might play an important role in the cyanidanol protection of CD-amplified CCl 4 toxicity. The present studies conducted over a time course of 0 to 120 hr after CCl 4 challenge describe sequential changes in hepatic [ 3H]thymidine incorporation into hepatocellular nuclear DNA, polyamines and related enzymes, and histomorphometry of liver sections from variously treated rats. Male Sprague-Dawley rats (125–150 g) were maintained on a control diet or on a diet contaminated with CD (10 ppm) for 15 days and/or pretreated with cyanidanol (250 mg/kg, ip) at 48, 24, and 2 hr befor a single ip injection of either a standard protocol dose (100 μl/kg) or a low dose (50 μl/kg, L) of CCl 4 on Day 16 of the dietary protocol. Cyanidanol pretreatment significantly stimulated the hepatic [ 3H]thymidine incorporation into hepatocellular nuclear DNA of control rats irrespective of CD pretreatment. Similarly, polyamine metabolism was altered favorably for cell division, although mitotic index (metaphase) was not increased. Cyanidanol-stimulated [ 3H]thymidine incorporation was highly suppressed in rats receiving the CD + CCl 4 standard dose combination treatment up to 36 hr, but after this time point a marked increase was observed. Hepatocellular regeneration, quantified histomorphometrically as volume density of cells in metaphase, was progressively increased in rats protected from CD + CCl 4 interaction by cyanidanol, starting at 36 hr and lasting until 72 hr. Favorably altered polyamine metabolism was evident from the stimulated ornithine decarboxylase, as well as from the stimulated interconversion of the higher polyamines to maintain increased concentration of putrescine. Challenge by the same dose of CCl 4 (100 μl/kg) to CD-pretreated rats not protected by cyanidanol failed to cause any increase in [ 3H]thymidine incorporation up to 36 hr and resulted in animal death starting at 36 hr. In the surviving rats, [ 3H]thymidine incorporation at 48 hr was increased, but was less than 50% of the increase observed in the cyanidanol group. In these rats, attenuation in the stimulation of cell division and insufficiently increaed putrescine levels were observed, which are consistent with the inadequate level of hepatocellular regeneration. With rats receiving CD + CCl 4(L) combination, the [ 3H]thymidine incorporation at 48 hr was less than 50% of the increase of cyanidanol-protected rats. Cyanidanol pretreatment to the CD + CCl 4 group of rats prevented the decrease in the hepatic DNA levels. CCl 4 challenge to control rats did not show any discernible alterations in the hepatic DNA levels. However, CCl 4 administration to CD-pretreated rats caused a significant and progressive decrease in hepatic DNA starting at 6 hr until animal death. Similarly, CD + CCl 4(L) rats showed a significant decrease in hepatic DNA at 24 and 48 hr, whereas, in the surviving rats a recovery in DNA levels was observed. These results suggest that the major event in the cyanidanol protection of CD-amplified CCl 4 toxicity is the stimulation of hepatocellular regeneration targeted for tissue repair.

Polyamine metabolism in some helminth parasites

Polyamine levels of some helminth parasites were analyzed by reverse phase HPLC of benzoyl derivatives. Setaria cervi, Acanthocheilonema viteae, Hymenolepis nana, H. diminuta, and Ascaridia galli contained higher levels of spermine than spermidine while in Ancylostoma ceylanicum and Nippostrongylus brasiliensis the spermidine levels were higher than spermine; putrescine was either absent or present in minor quantities. The enzymes of polyamine biosynthesis viz., ornithine decarboxylase, S-adenosyl methionine (SAM)-decarboxylase, and arginine decarboxylase were present in very low to negligible amounts in all the parasites examined. A. ceylanicum exhibited high activity of ornithine amino transferase (OAT) and catalyzed appreciable decarboxylation of ornithine. The ornithine decarboxylating activity of A. ceylanicum was localized in the participate fraction containing mitochondria, not inhibited by α-difluoromethyl ornithine, the specific inhibitor of ornithine decarboxylase (ODC), but inhibited in the presence of glutamate, suggesting the involvement of mitochondrial OAT rather than a true ODC in ornithine decarboxylation in this parasite. Significant activity of polyamine oxidase was also detected in helminth parasites. The absence of polyamine biosynthesizing enzymes in helminth parasites suggests their dependence on hosts for uptake and interconversion of polyamines, providing a potential target for chemotherapy.

Interconversion of polyamines in wild-type strains and mutants of yeasts and the effects of polyamines on their growth

Yeasts of wild-type strains, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans were shown to have the ability to form aminopropylcadaverine and aminopropylhomospermidine from cadaverine and homospermidine, respectively. A polyamine autotroph S. cerevisiae 179-5, which lacks ornithine decarboxylase, produced both aminopropylcadaverine and aminopropylhomospermidine, while another mutant S. cerevisiae Y 260 A, which lacks spermine synthase, formed only aminopropylcadaverine. Naturally-occurring triamines and tetraamines except norspermidine and norspermine stimulated the growth of S. cerevisiae 179-5. All the six aliphatic diamines with carbon chain length ranging from one to six were effective in activating the growth of S. cerevisiae 179-5, though all of them were not converted to either triamines or tetraamines.

Interconversion of polyamines in wild-type strains and mutants of yeasts and the effects of polyamines on their growth.

Yeasts of wild-type strains, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida albicans were shown to have the ability to form aminopropylcadaverine and aminopropylhomospermidine from cadaverine and homospermidine, respectively. A polyamine autotroph S. cerevisiae 179-5, which lacks ornithine decarboxylase, produced both aminopropylcadaverine and aminopropylhomospermidine, while another mutant S. cerevisiae Y 260 A, which lacks spermine synthase, formed only aminopropylcadaverine. Naturally-occurring triamines and tetraamines except norspermidine and norspermine stimulated the growth of S. cerevisiae 179-5. All the six aliphatic diamines with carbon chain length ranging from one to six were effective in activating the growth of S. cerevisiae 179-5, though all of them were not converted to either triamines or tetraamines.

Spermidine/spermine-N1-acetyltransferase activity in isoprenaline-stimulated rat salivary glands.

In parotid, sublingual and submaxillary glands stimulated by continuous intravenous infusion of isoprenaline at various doses for 3 h, the concentrations of the polyamines putrescine, spermidine, spermine and N1-acetylspermidine as well as the activities of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase were determined. Ornithine decarboxylase is the enzyme that catalyses the formation of putrescine from ornithine. The most striking effect was observed in parotid glands. Here, the beta-adrenergic agonist increased the activity of spermidine/spermine-N1-acetyltransferase markedly, but only slightly (if at all) that of ornithine decarboxylase. It also increased the concentrations of N1-acetylspermidine and putrescine markedly. The result obtained in this gland indicates interconversion of higher polyamines to putrescine.

Ascaris suum and Onchocerca volvulus: S-adenosylmethionine decarboxylase

Putrescine-dependent S-adenosylmethionine decarboxylase (EC 4.1.1.50) was demonstrated in Ascaris suum and Onchocerca volvulus; activation was found to be about fourfold by putrescine. Mg 2+ did not affect the enzyme activity. A. suum was taken as a model nematode and its S-adenosylmethionine decarboxylase was partially purified and characterized. The molecular weight was estimated to be 220,000. The apparent K m -value for adenosyl-methionine was determined to be 17 μM. Methylglyoxal bis(guanylhydrazone) and berenil competitively inhibited the enzyme activity; the apparent K i -values were found to be 0.24 μM and 0.11 μM, respectively. The dependence of filarial worms on uptake and interconversion of putrescine and polyamines as well as properties of the S-adenosylmethionine decarboxylase, different from the host enzyme, points to the polyamine metabolisms as a useful target for chemotherapy.

A specific and saturable spermine-binding component present in bovine testis membranes.

To determine if polyamine (PA)-binding components were present in testis membranes, a membrane fraction derived from homogenates of immature bovine testes was utilized in a radioligand assay. [14C]Spermine ([14C] Sp) bound to a spermine-binding component (SpBC) in a saturable manner and radioligand-binding data were analyzed and fit to a one-site model. Estimates of the binding constant (Kd = 7.2 +/- 1.2 X 10(-6) M) and of the concentration of sites (11.6 +/- 1.6 X 10(-6) M) were determined assuming a one-to-one stoichiometry. Metal cations (Ca+2, Mg+2, Mn+2 and Na+) as chloride salts had no effect on binding of [14C]Sp to SpBC when tested at concentrations up to 10 mM. Not all polyamines tested were effective competitors for [14C]Sp binding, which had an ID50 of 21.0 +/- 2.0 microM. The inhibitory potencies for putrescine and spermidine relative to spermine were 3.1 +/- 0.43 X 10(-4) and 0.1 +/- 0.01 (nmol/nmol) respectively, illustrating the specificity of SpBC binding. In addition, acetylation of polyamines, which is generally associated with degradation and interconversion of polyamines, resulted in a reduction of potency of 25.2 +/- 2.0-fold of spermine and 31.1 +/- 10.6-fold of spermidine in the SpBC radioligand assay. Binding of SpBC was decreased by tryptic digestion of the membrane fraction, suggesting that protein may be a part of the SpBC. Neuraminidase treatment had no effect on [14C]Sp binding but phospholipase-C digestion increased [14C]Sp binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential inhibition of histone and polyamine acetylases by multisubstrate analogues.

Mammalian cells contain a number of enzymes catalyzing the acetylation of polyamines and histones including an inducible spermidine/spermine N1-acetyltransferase which may play a key role in regulating the interconversion of polyamines [Matsui, I., Wiegand, L., & Pegg, A. E. (1981) J. Biol. Chem. 256, 2454-2459]. The present experiments were carried out in order to provide a method to distinguish this enzyme from other polyamine/histone acetylases and to test whether specific inhibitors of its activity could be obtained. Rabbit antiserum to homogeneous rat liver spermidine/spermine N1-acetyltransferase had no effect on the activity of a crude nuclear extract from rat liver, indicating that its spermidine acetylating capability is not related to the cytosolic spermidine/spermine N1-acetyltransferase induced by hepatotoxins. Potential multisubstrate analogues were prepared by attaching various polyamines to coenzyme A via an acetic acid linkage and tested as potential inhibitors of the acetylation of spermidine and histones. There was little difference in the potency of these polyamine derivatives as inhibitors of histone or spermidine acetylation by the crude nuclear extracts which appeared to contain at least two such activities, one inhibited completely by 20-30 microM and the other amounting to 50% of the total being unaffected by 100 microM. Spermidine/spermine N1-acetyltransferase was also inhibited by all the derivatives, but the potency toward this enzyme differed widely. The derivative from sym-norspermidine was a very strong inhibitor, giving 50% inhibition at 0.3 microM, and was more than 1 order of magnitude more active than the others.(ABSTRACT TRUNCATED AT 250 WORDS)