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.     

Polyamine degradation in foetal and adult bovine serum.

1. Using protein-separative chromatographic procedures and assays specific for putrescine oxidase and spermidine oxidase, adult bovine serum was found to contain a single polyamine-degrading enzyme with substrate preferences for spermidine and spermine. Apparent Km values for these substrates were approx. 40 microM. The apparent Km for putrescine was 2 mM. With spermidine as substrate, the Ki values for aminoguanidine (AM) and methylglyoxal bis(guanylhydrazone) (MGBG) were 70 microM and 20 microM respectively. 2. Bovine serum spermidine oxidase degraded spermine to spermidine to putrescine and N8-acetylspermidine to N-acetylputrescine. Acrolein was produced in all these reactions and recovered in quantities equivalent to H2O2 recovery. 3. Spermidine oxidase activity was present in foetal bovine serum, but increased markedly after birth to levels in adult serum that were almost 100 times the activity in foetal bovine serum. 4. Putrescine oxidase, shown to be a separate enzyme from bovine serum spermidine oxidase, was present in foetal bovine serum but absent from bovine serum after birth. This enzyme displayed an apparent Km for putrescine of 2.6 microM. The enzyme was inhibited by AM and MGBG with Ki values of 20 nM. Putrescine, cadaverine and 1,3-diaminopropane proved excellent substrates for the enzyme compared with spermidine and spermine, and N-acetylputrescine was a superior substrate to N1- or N8-acetylspermidine.     

Glucocorticoid Regulation of Spermidine Acetylation in the Rat Brain

The effect of glucocorticoids on polyamine metabolism has been elucidated further by measuring putrescine, spermidine, and spermine levels as well as ornithine decarboxylase, S-adenosylmethionine decarboxylase, and N1-acetylspermidine transferase activities in the hippocampus, cerebellar cortex, vermis, and deep nuclei of adrenalectomized rats. At 6 h after corticosterone or dexamethasone administration, the specific activities of ornithine decarboxylase and N1-acetylspermidine transferase showed the greatest increases in all brain tissues examined, and at 12 h, S-adenosylmethionine decarboxylase activity was not increased significantly. The hippocampus and cerebellar regions displayed different responses to corticosterone and dexamethasone, corresponding to the distribution of glucocorticoid and mineralocorticoid receptors. Corticosterone and dexamethasone increased ornithine decarboxylase and N1-acetylspermidine transferase activities in a dose-dependent manner, with dexamethasone being more active than corticosterone in all tissues. However, estradiol, progesterone, testosterone, and aldosterone were only active at doses greater than 5 mg/kg. The great increases in ornithine decarboxylase and N1-acetylspermidine transferase activities were accompanied by a marked increase in putrescine level and a small decrease in spermidine level. Our data confirm that the hippocampus and cerebellum are glucocorticoid target tissues and suggest that the increase in the content of putrescine, following acute treatment with glucocorticoids, is dependent on ornithine decarboxylase as well as N1-acetylspermidine transferase induction.     

Yeast Fms1 is a FAD-utilizing polyamine oxidase

In this report we show that recombinant Saccharomyces cerevisiae Fms1 protein is a polyamine oxidase that binds FAD with an FAD:Fms1 stoichiometry of 1:1. Biochemical characterization of Fms1 shows that it can oxidize spermine, N(1)-acetylspermine, N(1)-acetylspermidine, and N(8)-acetylspermidine, but not spermidine. The products of spermine oxidation are spermidine and 3-aminopropanal. A kinetic analysis revealed that spermine, N(1)-acetylspermine, and N(1)-acetylspermidine are oxidized with similar efficiencies, while N(8)-acetylspermidine is a poor substrate. The data support a previous report, suggesting that Fms1 is responsible for the production of beta-alanine from spermine for the synthesis of pantothenic acid.     

Effects of dexamethasone on spermidine N1-acetyltransferase and ornithine 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.     

Enhancement of ouabain and calcium ionophore A23187 of outward transport of polyamines from lymphocytes

Human lymphocytes in culture loaded with radioactive polyamines slowly release radioactivity into the medium. N1-Acetylspermidine is mostly released from spermidine and spermine. Both ouabain and calcium ionophore A23187 increase the outward transport, but by different mechanisms. Ouabain inhibits the acetylation of spermidine, and free spermidine is released, whereas A23187 increases both acetylation of spermidine and the efflux of N1-acetylspermidine.     

Calf liver nuclear N-acetyltransferases. Purification and properties of two enzymes with both spermidine acetyltransferase and histone acetyltransferase activities.

Calf liver contains two nuclear N-acetyltransferases which are separated by chromatography on hydroxylapatite. Both acetyltransferase A and acetyltransferase B will transfer acetate from acetyl-CoA to either histone or spermidine. The same protein catalyzes the reaction with both substrates; this is shown by a constant ratio of spermidine to histone activity over a 5,000-fold purification and identical heat denaturation kinetics for both spermidine and histone acetyltransferase activity with each enzyme. Histone is preferentially acetylated when both acceptors are present. Both enzymes preferentially acetylate polyamines (spermidine, spermine, and diaminodipropylamine) to diamines. Acetyltransferase A acetylates histones in the order: whole histone greater than H4 greater than H2A greater than H3 greater than H2B greater than H1; acetyltransferase B in the order: whole histone greater than H4 = H3 greater than H2A greater than H2B greater than H1. Michaelis constants are 2 X 10(-4)M for spermidine and 9 X 10(-6)M for acetyl-CoA. Acetyltransferase A has a molecular weight of 150,000; acetyltransferase B 175,000. Both enzymes are strongly inhibited by p-chloromercuribenzoate and weakly inhibited by EDTA.     

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.     

Acetylated polyamines as substrates for human pregnancy serum diamine oxidase

Human pregnancy serum diamine oxidase was purified 50 fold and tested for activity with a variety of substrates. Putrescine, spermidine, spermine, N-acetylputrescine, N⁸-acetylspermidine, and N¹-acetylspermidine were acceptable substrates for the enzyme, which exhibited greatest activity against N¹-acetylspermidine.     

Guide molecule-driven stereospecific degradation of alpha-methylpolyamines by polyamine oxidase

FAD-dependent polyamine oxidase (PAO; EC 1.5.3.11) is one of the key enzymes in the catabolism of polyamines spermidine and spermine. The natural substrates for the enzyme are N1-acetylspermidine, N1-acetylspermine, and N1,N12-diacetylspermine. Here we report that PAO, which normally metabolizes achiral substrates, oxidized (R)-isomer of 1-amino-8-acetamido-5-azanonane and N1-acetylspermidine as efficiently while (S)-1-amino-8-acetamido-5-azanonane was a much less preferred substrate. It has been shown that in the presence of certain aldehydes, the substrate specificity of PAO and the kinetics of the reaction are changed to favor spermine and spermidine as substrates. Therefore, we examined the effect of several aldehydes on the ability of PAO to oxidize different enantiomers of alpha-methylated polyamines. PAO supplemented with benzaldehyde predominantly catalyzed the cleavage of (R)-isomer of alpha-methylspermidine, whereas in the presence of pyridoxal the (S)-alpha-methylspermidine was preferred. PAO displayed the same stereospecificity with both singly and doubly alpha-methylated spermine derivatives when supplemented with the same aldehydes. Structurally related ketones proved to be ineffective. This is the first time that the stereospecificity of FAD-dependent oxidase has been successfully regulated by changing the supplementary aldehyde. These findings might facilitate the chemical regulation of stereospecificity of the enzymes.     

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.     

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.     

Agmatine modulates polyamine content in hepatocytes by inducing spermidine/spermine acetyltransferase.

Agmatine has been proposed as the physiological ligand for the imidazoline receptors. It is not known whether it is also involved in the homoeostasis of intracellular polyamine content. To show whether this is the case, we have studied the effect of agmatine on rat liver cells, under both periportal and perivenous conditions. It is shown that agmatine modulates intracellular polyamine content through its effect on the synthesis of the limiting enzyme of the interconversion pathway, spermidine/spermine acetyltransferase (SSAT). Increased SSAT activity is accompanied by depletion of spermidine and spermine, and accumulation of putrescine and N1-acetylspermidine. Immunoblotting with a specific polyclonal antiserum confirms the induction. At the same time S-adenosylmethionine decarboxylase activity is significantly increased, while ornithine decarboxylase (ODC) activity and the rate of spermidine uptake are reduced. This is not due to an effect on ODC antizyme, which is not significantly changed. All these modifications are observed in HTC cells also, where they are accompanied by a decrease in proliferation rate. SSAT is also induced by low oxygen tension which mimics perivenous conditions. The effect is synergic with that promoted by agmatine.     

Studies of the specificity and kinetics of rat liver spermidine/spermine N1-acetyltransferase.

The substrate specificity and kinetic mechanism of spermidine N1-acetyltransferase from rat liver was investigated using a highly purified (18 000-fold) preparation from the livers of rats in which the enzyme was induced by treatment with carbon tetrachloride (1.5 ml/kg body wt. 6h before death). The enzyme catalysed the acetylation of spermidine, spermine, sym-norspermidine, sym-norspermine, N-(3-aminopropyl)-cadaverine, N1-acetylspermine, 3,3'-diamino-N-methyldipropylamine and 1,3-diaminopropane, but was inactive with putrescine, cadaverine, sym-homospermidine and N1-acetylspermidine. These results suggest that the enzyme is highly specific for the acetylation of a primary amino group that is separated by a three-carbon aliphatic chain from another nitrogen atom (i.e. the substrates are of the type H2N[CH2]3NHR). The maximal rates of acetylation of 1,3-diaminopropane and 3,3'-diamino-N-methyldipropylamine were much lower than the maximal rates with spermidine or sym-norspermidine as substrates, suggesting a preference for a secondary amino group bearing the aminopropyl group that is acetylated. The best substrates for acetylation were sym-norspermidine and sym-norspermine, which had Km values of about 10 micrograms and Vmax. values of about 2 mumol of product/min per mg of enzyme compared with Km of 130 microM and Vmax. of 1.3 mumol/min per mg for spermidine. N1-Acetylspermidine (the product of the reaction) and N8-acetylspermidine were weak inhibitors and were competitive with spermidine, having Ki values of about 6.6 mM and 0.4 mM respectively. N1-Acetylspermidine was a non-competitive inhibitor with respect to acetyl-CoA. CoA was also inhibitory to the reaction, showing non-competitive kinetics when either [acetyl-CoA] or [spermidine] was varied. These results suggest that the reaction occurs via an ordered Bi Bi mechanism in which spermidine binds first and N1-acetyl-spermidine is the final product to be released.     

The occurrence, subcellular localization and partial purification of diamine acetyltransferase in the yeast Candida boidinii grown on spermidine or putrescine as sole nitrogen source

The yeast Candida boidinii when grown on spermidine, diaminopropane, putrescine or cadaverine as sole nitrogen source contains an N-acetyltransferase capable of acetylating the primary amino groups of spermine, spermidine, acetylspermidines, acetylputrescine and alpha, omega-diaminoalkanes. In the case of spermidine, the products were N1-acetylspermidine and N8-acetylspermidine in the ratio 50:45 with traces of other unidentified products. The enzyme was partially purified and the stoichiometry determined, together with apparent Km and V values for a number of substrates. The pH optimum was about 8.8 for putrescine and 9.3 for spermidine. The unstable enzyme was partially stabilized by 10% (v/v) glycerol or bovine serum albumin (5 mg/ml). The kinetic parameters were determined with putrescine as substrate and the mechanism shown to be of the sequential type. The enzyme was shown to be located in the mitochondria of C. boidinii, in contrast to mammalian N-acetyltransferases. The enzyme was found in a number of other yeast species when grown on spermidine or putrescine, but was only present in those species that had previously been found to contain polyamine oxidase. It is suggested that in C. boidinii, as in mammals, acetylation of spermidine and putrescine must precede their catabolism.     

S-Adenosylmethionine Decarboxylase Levels in Rat Retina During Postnatal Development

Abstract: S -Adenosylmethionine decarboxylase from rat retina is similar to that isolated from other rat tissues with regard to kinetic parameters. pH optimum, putrescine requirement, and sensitivity to spermine. The enzymic activity increases during the first 7 days of postnatal life but decreases until the 20th day. After this period AdoMet decarboxylase activity increases, to reach the highest values at the 90th day. This behavior suggests that such enzymic activity is responsible for spermidine and spermine levels in rat retina and that a high content of retinal spermine might have a role in the photoreceptor outer segment renewal.     

Role of protein kinase C in phytohemagglutinin-stimulated induction of spermidine/spermine N1-acetyltransferase

We studied the involvement of protein kinase C in the induction of spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme of polyamine degradation, in bovine lymphocytes. When phytohemagglutinin (PHA) and H-7, a protein kinase inhibitor, were added simultaneously to lymphocyte cultures, the elevation caused by PHA of spermidine/spermine N1-acetyltransferase activity at 24 h after administration was reduced. In cells treated with a lower concentration of PHA, the acetyltransferase activity was enhanced with 12-o-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, and reached the level of cells with a higher concentration of PHA. PHA did not cause maximum induction of the enzyme in cells treated with 160 ng/ml TPA. The induction of this acetyltransferase with PHA is probably mediated by protein kinase C.     

Characterization of human spermidine/spermine N1-acetyltransferase purified from cultured melanoma cells

Extreme inducibility of spermidine/spermine acetyltransferase (SSAT) by bis-ethyl derivatives of spermine in human large cell lung carcinoma and melanoma cells has prompted biochemical characterization of the purified enzyme. Treatment of human MALME-3 melanoma cells with 10 microM N1,N11-bis(ethyl)norspermine (BENSPM) for 48-72 h increased SSAT activity by some 1000- to 4000-fold and enabled purification of the enzyme by established procedures--binding on immobilized spermine and elution with spermine followed by binding on Matrex Blue A and elution with coenzyme A. The enzyme showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a single subunit species and molecular weight of approximately 20,300 Da. By gel permeation chromatography, the holoenzyme was found to have a molecular weight of 80,000 Da, suggesting a total of four identical subunits. Purified SSAT had a specific activity of 285 mumol/min/mg for spermidine and Km values of 5.9 microM for acetylcoenzyme A, 55 microM for spermidine, 5 microM for spermine, 36 microM for N1-acetylspermine, 1.6 microM for norspermidine, and 4 microM for norspermine. Homologs of BENSPM were found to be competitive inhibitors of spermidine acetylation, with Ki values of 0.8 microM for BENSPM, 1.9 microM for N1,N12-bis-(ethyl)spermine and 17 microM for N1,N14-bis-(ethyl)-homospermine. Correlation of these values with the relative abilities of the homologs to increase SSAT in intact cells suggests that formation of an enzyme inhibitor complex may play a contributing role in enzyme induction.     

Polyamines and ornithine decarboxylase activity in the developing rat retina

The behaviour of ornithine decarboxylase activity and the changes of polyamine (spermidine and spermine) and putrescine concentrations in the rat retina during the postnatal development have been studied.In the first 12 days of life, when cellular division first and then cellular differentiation are known to occur in rat retina, polyamine concentrations and enzymic activity rise to and maintain their maximum values.After 12 days of life, putrescine and polyamine retinal levels are drastically reduced, and adult values are already reached at the age of 16 days. The adult level of spermine is six to seven times greater than the low values obtained for both putrescine and spermidine. This relatively high content of spermine could be related to the mechanism of perpetual renewal of photoreceptor outer segments.