Regulation of spermidine/spermine N1-acetyltransferase in L6 cells by polyamines and related compounds.

Exposure of rat L6 cells in culture to exogenous polyamines led to a very large increase in the activity of spermidine/spermine N1-acetyltransferase. Spermine was more potent than spermidine in bringing about this increase, but in both cases the elevated acetyltransferase activity increased the cellular conversion of spermidine into putrescine. The N1-acetyltransferase turned over very rapidly in the L6 cells, with a half-life of 9 min after spermidine and 18 min after spermine. A wide variety of synthetic polyamine analogues also brought about a substantial induction of spermidine/spermine N1-acetyltransferase activity. These included sym-norspermidine, sym-norspermine, sym-homospermidine, N4-substituted spermidine derivatives, 1,3,6-triaminohexane, 1,4,7-triaminoheptane and deoxyspergualin, which were comparable with spermidine in their potency, and N1N8-bis(ethyl)spermidine, N1N9-bis(ethyl)homospermidine, methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone) and 1,1'-[(methylethanediylidene)dinitrilo]bis(3-amino-guanidine ), which were even more active than spermidine. It is suggested that these polyamine analogues may bring about a decrease in cellular polyamines not only by inhibiting biosynthesis but by stimulating the degradation of spermidine into putrescine.     

Effect of N1,N12-bis(ethyl)spermine and related compounds on growth and polyamine acetylation, content, and excretion in human colon tumor cells

Exposure of human colon tumor (HT 29 cells) to N1,N12-bis(ethyl)spermine and analogs produced a rapid loss of intracellular polyamines. This loss was brought about predominantly by an increased excretion of spermidine. N1,N11-Bis(ethyl)norspermine and N1,N12-Bis(ethyl)spermine were potent inducers of spermidine/spermine N1-acetyltransferase, and this induction facilitated the efflux of polyamines by enhancing the conversion of spermine into spermidine. N1,N14-Bis(ethyl)homospermine, which did not induce spermidine/spermine N1-acetyltransferase, also caused the loss of spermidine from the cell but was less effective in bringing about the decline in intracellular spermine. These results indicate that cellular polyamine levels can be regulated by excretion of spermidine and that the bis(ethyl)spermine derivatives deplete intracellular polyamine content by interference with this process.     

Variations in endogenous polyamine content of maize calli obtained from zygotic and androgenetic embryos

A comparative study of polyamine (putrescine, spermidine and spermine) levels was conducted with maize calli originating from a) immature embryos and b) pollen embryos capable of plant regeneration. The differences observed in the studied parameters of the two kinds of calluses are related to their cellular origin and to their regeneration capacity. Moreover, only the calluses proceeding from immature embryos differentiated into preembryogenic structures, which eventually developed into plants. Although total polyamine levels in pollenderived calluses were significantly higher than those from immature embryos, spermidine and spermine were the predominant polyamines in both culture types. Furthermore, polyamine fractions of these calluses also showed differences. All these phenomena may be related with the differences observed in the callus embryogenic response. These findings may be useful in understanding the implication of polyaminesin embryogenetic processes.Abbreviations IEC immature-embryo calluses - PAs polyamines - PEC pollen-embryo calluses - PH insoluble conjugated PA fraction - Put putrescine - S free PA fraction - SH soluble conjugated PA fraction - Spd spermidine - Spm spermine 2,4d-2,4 dichlorophenoxyacetic acid     

Bis(glutathionyl)spermine and other novel trypanothione analogues in Trypanosoma cruzi

Trypanosomatids differ from other cells in their ability to conjugate glutathione with the polyamine spermidine to form the antioxidant metabolite trypanothione (N1,N8-bis(glutathionyl)spermidine). In Trypanosoma cruzi, trypanothione is synthesized by an unusual trypanothione synthetase/amidase (TcTryS) that forms both glutathionylspermidine and trypanothione. Because T. cruzi is unable to synthesize putrescine and is dependent on uptake of exogenous polyamines by high affinity transporters, synthesis of trypanothione may be circumstantially limited by lack of spermidine. Here, we show that the parasite is able to circumvent the potential shortage of spermidine by conjugating glutathione with other physiological polyamine substrates from exogenous sources (spermine, N8-acetylspermidine, and N-acetylspermine). Novel thiols were purified from epimastigotes, and structures were determined by matrix-assisted laser desorption ionization time-of-flight analysis to be N1,N12-bis(glutathionyl)spermine, N1-glutathionyl-N8-acetylspermidine, and N1-glutathionyl-N12-acetylspermine, respectively. Structures were confirmed by enzymatic synthesis with recombinant TcTryS, which catalyzes formation of these compounds with kinetic parameters equivalent to or better than those of spermidine. Despite containing similar amounts of spermine and spermidine, the epimastigotes, trypomastigotes, and amastigotes of T. cruzi preferentially synthesized trypanothione. Bis(glutathionyl)spermine disulfide is a physiological substrate of recombinant trypanothione reductase, comparable to trypanothione and homotrypanothione disulfides. The broad substrate specificity of TcTryS could be exploited in the design of polyamine-based inhibitors of trypanothione metabolism.     

Polyamine distributions in thermophilic eubacteria belonging to Thermus and Acidothermus

Triamines such as norspermidine, spermidine, and homospermidine and tetraamines such as norspermine, spermine, thermospermine, and aminopropylhomospermidine were found to be distributed ubiquitously in the eight extremely thermophilic (growing at 70 degrees C) Thermus species tested. Three linear pentaamine (caldopentamine, homocaldopentamine, and thermopentamine), two linear hexaamines (caldohexamine and homocaldohexamine), two tertiary branched tetraamines (N4-aminopropylnorspermidine and N4-aminopropyl-spermidine), and quaternary branched pentaamines such as N4-bis(aminopropyl)norspermidine and N4-bis(aminopropyl)spermidine were detected in T. thermophilus HB8, T. filiformis Wai33 A1, T. flavus AT-62, and T. caldophilus GK24. The linear hexaamines and branched polyamines were absent in T. aquaticus YT-1, T. sp. X-1, T. sp. T2, and T. sp. T351, in which linear pentaamines were minor components. Moderately thermophilic Thermus ruber and Thermus sp. K-2 contained putrescine, spermidine, norspermidine, homospermidine, spermine, norspermine, thermospermine, and aminopropylhomospermidine. No pentaamines, hexaamines, or branched polyamines were found in these two moderately thermophilic Thermus species. On the other hand, moderately thermophilic, acidophilic Acidothermus cellulolyticus was devoid of all the polyamines.     

gamma-Glutamylamine derivatives in isolated rat hepatocyte proteins.

Freshly isolated rat hepatocytes were found to contain a 3-fold higher level of putrescine than perfused liver. The bulk of this diamine was recovered in the acid-insoluble fraction of the cell. In order to determine the nature of the amine binding, the levels of gamma-glutamylamine derivatives were measured. The method used involves exhaustive proteolytic digestion of the acid-insoluble fraction of hepatocytes, followed by ion-exchange chromatography. For N1-(gamma-glutamyl)putrescine, a combined ion-exchange chromatographic and reverse-phase h.p.l.c. procedure was adopted. This allowed for the direct detection of less than 50 pmol of this derivative in enzymic hydrolysates. Several of the gamma-glutamylamines reported previously [Beninati, Piacentini, Argento-Ceru', Russo-Caia & Autuori (1985) Biochim. Biophys. Acta 841, 120-126] in the whole organ were found in the isolated liver cells. The elevated level of N1-(gamma-glutamyl)putrescine and the absence of bis-(gamma-glutamyl)spermine was noteworthy. The results suggest that, in rat hepatocytes, both polyamine-dependent post-translational modification of some proteins and cross-linking between proteins involving the glutamine and lysine residues occurs.     

Acrolein produced from polyamines as one of the uraemic toxins

It is well known that the addition of spermine or spermidine to culture medium containing ruminant serum inhibits cellular proliferation. This effect is caused by the products of oxidation of polyamines that are generated by serum amine oxidase. Among the products, we found that acrolein is a major toxic compound produced from spermine and spermidine by amine oxidase. We then analysed the level of polyamines (putrescine, spermidine and spermine) and amine oxidase activity in plasma of patients with chronic renal failure. It was found that the levels of putrescine and the amine oxidase activity were increased, whereas spermidine and spermine were decreased in plasma of patients with chronic renal failure. The levels of free and protein-conjugated acrolein were also increased in plasma of patients with chronic renal failure. An increase in putrescine, amine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. These results suggest that acrolein is produced during the early stage of nephritis through kidney damage and also during uraemia through accumulation of polyamines in blood due to the decrease in their excretion into urine.     

N1-monoacetylation abolishes the inhibitory effect of spermine and spermidine in the reticulocyte lysate translation system

At optimum magnesium, the translation of rat heart mRNA in the nuclease treated rabbit reticulocyte lysate system was inhibited by low concentrations of spermidine or spermine but not of putrescine. Spermidine and spermine cause a general reduction in the translation of all the heart mRNAs since no differential effects were observed when the translation products were examined by gel electrophoresis. Spermine was a five times more potent inhibitor than spermidine but no inhibition was obtained with N1-acetylspermidine or N1-acetylspermine. Since analyses of endogenous polyamines demonstrate that the inhibitory concentrations of spermine could be obtained by converting a small fraction of the endogenous spermidine to spermine, these results indicate that interconversions of the polyamines might be a sensitive regulatory mechanism for protein synthesis.     

Presence of di- and polyamines covalently bound to protein in rat liver

Acid hydrolysis of trichloroacetic acid precipitate from rat tissue (liver, kidney and testis) homogenate released significant amounts of acid-insoluble putrescine, spermidine and spermine. Following incubation of liver homogenate with [1,4-¹⁴C]putrescine, 1.4% of total radioactivity and 1.0% of labelled diamine were recovered in the acid-insoluble fraction. Exhaustive digestion of acid-precipitable material with proteinases (Pronase, aminopeptidase M, carboxipeptidase A, B and Y) revealed the presence of di- and polyamines and of N¹-(γ-glutamyl)spermidine, N¹-(γ-glutamyl)sperminine and N¹, N¹²-bis(γ-glutamyl)spermine. These derivatives were identified both by chromatographic analysis and by enzymatic digestion with purified γ-glutamylamine cyclotransferase. The finding of di- and polyamine γ-glutamyl derivatives in the proteinase-digested acid-insoluble fraction of homogenate may be considered as a proof of the in vivo transglutaminase-catalyzed binding of polyamines to proteins. This evidence suggests that di- and polyamines might have an important role in mammalian tissues through covalent binding to proteins by either one or both the primary amino groups.     

Excretion of acetylated and free polyamines by polyamine depleted Chinese hamster ovary cells

1. Cultured Chinese hamster ovary cells (CHO) and their ornithine decarboxylase deficient mutant cells (C55.7) were found to excrete small amounts of N8-acetylspermidine and free polyamines, putrescine and spermidine into the culture medium. 2. The concentration of N8-acetylspermidine in the control cells was 2-3% of that of spermidine. In the medium, however, the amount of N8-acetylspermidine was about 2-fold that of spermidine and 2- to 3-fold higher than the intracellular amount. N1-acetylspermidine or acetylated spermine were never detected in the cells or in the media. 3. Confluent CHO cells treated with 2 mM difluoromethylornithine stopped the excretion when the intracellular spermidine concentration had decreased to 20% of control while there was no decrease in spermine concentration. At low cell density, neither polyamine depleted CHO cells nor the C55.7 cells excreted any polyamines into the culture media.     

Targeted disruption of spermidine/spermine N1-acetyltransferase gene in mouse embryonic stem cells. Effects on polyamine homeostasis and sensitivity to polyamine analogues

We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N(1),N(11)-diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C(14)]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N(1)-acetylspermidine and putrescine were readily detectable in N(1),N(11)-diethylnorspermine-exposed wild-type cells but not in SSAT-deficient cells. Similar experiments with [C(14)]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.     

Determination of polyamines in digestive and reproductive tissues of adult Asterias vulgaris (Echinodermata: Asteroidea)

1. The polyamines spermine, spermidine and putrescine were extracted from tissues of Asterias vulgaris and quantitated using thin layer chromatography. 2. In the pyloric caeca, mean (+/- SE) levels of spermine, spermidine and putrescine were 138(15), 86(24) and 415(77) nmol/g wet wt tissue, respectively. In the testes, levels were 95(12), 13(6) and less than 6 nmol/g wet wt. In the ovaries, levels were 105(9), 11(0.8) and 15(8) nmol/g wet wt. 3. High levels of polyamines in the pyloric caeca may be related to secretion or excretion in this tissue. 4. We hypothesize that polyamines will be important in the regulation of cellular activity in these tissues during the annual reproductive cycle.     

Polyamine metabolism of filaria and allied parasites

Putrescine and the polyamines spermidine and spermine occur both in prokaroytes and in eukaryotes where they seem intimately involved in regulatory processes of cellular growth and differentiation. They seem to play an important role related to the biosynthesis of nucleic acids and proteins, although at the molecular level their precise function remains unclear. In general, prokaryotes utilize putrescine and spermidine while eukaryotes tend to have higher concentrations of spermidine and spermine compared to putrescine(1-3.) Differences in polyamine metabolism between parasites and their hosts suggest several potential targets for chemotherapeutic attack As Rolf Walter discusses here, such approaches have already been exploited for African trypanosomes and also offer some leads for the chemotherapy of helminth infections.     

Polyamine oxidase and tissue transglutaminase activation in rat small intestine by polyamines.

Polyamine degradation was studied in the small intestine from rats fed on a polyamine-supplemented diet. Lactalbumin diet was given to Hooded-Lister rats, with or without 5 mg rat(-1) day(-1) of putrescine or spermidine for 5 days. Polyamine oxidase activity increased with putrescine and spermidine in the diet, whereas spermidine/spermine N(1)-acetyltransferase and diamine oxidase activities were unchanged. We also studied the calcium-dependent and -independent tissue transglutaminase activities, since they can modulate intestinal polyamine levels. Both types of enzymes increased in the cytosolic fraction after putrescine (about 65%) or spermidine (80-100%). Our results indicate that exogenous polyamines stimulate intestinal polyamine oxidase and tissue transglutaminase activities, probably to prevent polyamine accumulation, when other pathways of polyamine catabolism (acetylation and terminal catabolism) are not activated.     

Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.

Treatment of rats with spermidine, spermine or sym-norspermidine led to a substantial induction of spermidine/spermine N1-acetyltransferase activity in liver, kidney and lung. The increase in this enzyme, which was determined independently of other acetylases by using a specific antiserum, accounted for all of the increased acetylase activity in extracts from rats treated with these polyamines. Spermine was the most active inducer, and the greatest effect was seen in liver. Liver spermidine/spermine N1-acetyltransferase activity was increased about 300-fold within 6 h of treatment with 0.3 mmol/kg doses of spermine; activity in kidney increased 30-fold and activity in the lung 15-fold under these conditions. The increased spermidine/spermine N1-acetyltransferase activity led to a large increase in the liver putrescine content and a decline in spermidine. These changes are due to the oxidation by polyamine oxidase of the N1-acetylspermidine formed by the acetyltransferase. Our results indicated that spermidine was the preferred substrate in vivo of the acetylase/oxidase pathway for the conversion of the higher polyamines into putrescine. The induction of the spermidine/spermine N1-acetyltransferase by polyamines may provide a mechanism by which excess polyamines can be removed.     

Polyamines decrease Ca(2+) sensitivity of tension and increase rates of activation in skinned cardiac myocytes

Owing in part to their interactions with membrane proteins, polyamines (e.g., spermine, spermidine, and putrescine) have been identified as potential modulators of membrane excitability and Ca(2+) homeostasis in cardiac myocytes. To investigate whether polyamines also affect cardiac myofilament proteins, we assessed the effects of polyamines on contractility using rat myocytes and trabeculae that had been permeabilized with Triton X-100. Spermine, spermidine, and putrescine reversibly increased the [Ca(2+)] required for half-maximal tension (i.e., right-shifted tension pCa curves), with the following order of efficacy: spermine (+4) > spermidine (+3) > putrescine (+2). However, synthetic analogs that differed from spermine in charge distribution were not as effective as spermine in altering isometric tension. None of the polyamines had a significant effect on maximal tension, except at high concentrations. After flash photolysis of DM-Nitrophen (a caged Ca(2+) chelator), spermine accelerated the rate of tension development at low and intermediate but not high [Ca(2+)]. These results indicate that polyamines, especially spermine, interact with myofilament proteins to reduce apparent Ca(2+) binding affinity and speed cross-bridge cycling kinetics at submaximal [Ca(2+)].     

Purification by affinity chromatography and characterization of porcine liver cytoplasmic polyamine oxidase

1. Polyamine oxidase was purified from the soluble fraction of porcine liver by more than 70,000-fold to electrophoretic homogeneity using N8-acetylspermidine-Sepharose 4B affinity chromatography. 2. The molecular weight and isoelectric point of this enzyme were 62,000 and pH 4.5, respectively. 3. Optimal pH for the catalytic activity was close to 10.0. 4. The enzyme activity was enhanced by 5 mM dithiothreitol or 5 mM benzaldehyde. 5. Preferential substrates for this cytoplasmic PAO were N1-acetylspermine, N1-acetylspermidine and spermine. 6. Spermidine was not virtually the substrate for this enzyme. 7. The present results suggested the physiological roles of cytoplasmic PAO, being coupled with the reaction of spermidine/spermine N1-acetyltransferase, in recycling the cellular polyamines to putrescine.     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Investigation of polyamine metabolism by high-performance liquid chromatographic and gas chromatographic profiling methods

Measurements of polyamines, polyamine conjugates and their metabolites in tissues, cells and extracellular fluids are used in biochemistry, (micro)biology, oncology and parasitology. Decarboxylation of ornithine yields putrescine. Aminopropylation of putrescine yields spermidine, and aminopropylation of spermidine yields spermine. Spermidine and spermine are retroconverted to putrescine and spermidine, respectively, by initial N-acetylation and subsequent polyamine oxidation. The intermediate N-acetylputrescine, N¹-acetylspermidine and N⁸-acetylspermidine are the major urinary N-acetylpolyamines. Polyamines and N-acetylpolyamines are terminally degraded to non-α-amino acid metabolites by oxidative deamination and aldehyde dehydrogenation. Chromatography with on-line detection is the most commonly applied profiling method for polyamines, N-acetylpolyamines and their non-α-amino acid metabolites. Cation-exchange and reversed-phase high-performance liquid chromatography require pre- or post-column derivatisation, followed by UV-Vis spectrophotometric or fluorimetric detection. Isolation and derivatisation precedes gas chromatography with flame-ionisation, nitrogen-phosphorus, electron-capture or mass spectrometric detection. High-performance liquid chromatography and gas chromatography of polyamines are not competitive techniques, but rather supplementary.