Fluorinated analogues of spermidine as substrates of spermine synthase

A series of mono- and geminal difluorinated analogues of spermidine (4-azaoctane-1,8-diamine) have been tested as potential substrates of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase (EC 2.5.1.22). Substitution of the hydrogen atoms of the methylene group at position 7 by one or two fluorine atoms decreases 8-fold and 160-fold the apparent Km values for the HTC cell enzyme respectively. Similarly, the Km values of 7-monofluoro and 7,7-difluorospermidine for the pure bovine enzyme are reduced 8-fold and 100-fold respectively, in comparison with spermidine. Di-, but not monofluoro substitution, in the 6-position causes a 5-fold reduction in the affinity for the HTC cell enzyme. Gem-fluorine substitution in the 2-position abolishes substrate capability. In addition to their high affinity for spermine synthase, 7-monofluorospermidine and 7,7-difluorospermidine cause substrate inhibition. This phenomenon, which is more pronounced in the case of the difluorinated analogues is pH-dependent. These enzymatic findings are discussed with regard to the protonation sites of the spermidine analogues, determined by potentiometric titration, which vary as a function of the number and position of the fluorine substituents relative to the basic amino groups.     

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.     

Studies of the acetyl-CoA-binding site of rat liver spermidine/spermine N1-acetyltransferase.

Rat liver spermidine/spermine N1-acetyltransferase was found to be strongly inhibited by the dyes Cibacron F3GA, Coomassie Brilliant Blue and Congo Red. Inhibition was competitive with respect to acetyl-CoA and Ki values of 0.7 microM and 52 microM were determined for Cibacron F3GA and Coomassie Brilliant Blue respectively. The enzyme was strongly retained by columns of Affi-Gel Blue, which contains Cibacron F3GA linked to agarose. It was not eluted from this adsorbent in the presence of 10 mM-spermidine/0.5 M-NaCl/50 mM-Tris/HCl, pH 7.5, but was released by 1 mM-CoA in 10 mM-spermidine/50 mM-Tris/HCl, pH 7.5. These results are consistent with the presence in the enzyme of a dinucleotide fold that binds acetyl CoA and has a high affinity for Cibacron F3GA. The spermidine/spermine N1-acetyltransferase was irreversibly inactivated by exposure to butane-2,3-dione in sodium borate, pH 7.8, or by exposure to phenylglyoxal or camphorquinone-10-sulphonic acid. All of these reagents are known to interact with arginine residues in proteins under the conditions in which they inactivated the acetyltransferase. Inactivation was prevented by the presence of acetyl-CoA or CoA, but to a lesser extent by 3'-dephospho-CoA and not at all by NAD or adenosine. This protection suggests that an arginine residue at the active site is involved in the binding of the acetyl-CoA substrate. Treatment of the assay mixture but not the spermidine N1-acetyltransferase with alkaline phosphatase prevented the reaction taking place. This suggests that the apparent loss of enzyme activity in response to alkaline phosphatase reported by Matsui, Otani, Kamei & Morisawa [(1982) FEBS Lett. 150, 211-213] is due to dephosphorylation of the acetyl-CoA substrate and that the 3'-phosphate group is essential for activity.     

Acetylation of spermidine and spermine by rat liver and kidney chromatin

The in vitro enzymatic acetylation of the polyamines, spermidine and spermine, is described. The reaction is catalyzed by chromatin preparations from rat liver and kidney and is dependent on acetyl-CoA. Spermidine, spermine, and putrescine are each converted to the corresponding monoacetyl derivatives. s_0.5 values of 0.5 ± 0.1, 1.0 ± 0.1, and 2.6 ± 0.7 mm (mean ± standard deviation) were obtained for spermidine, spermine, and putrescine, respectively. These values for s_0.5 are similar to the concentrations of polyamines reported for tissues, and therefore, suggest the occurrence of polyamine acetylation in vivo. Evidence is also presented for the metabolism of acetylated polyamines by the 100,000g supernatant fraction of rat liver. The physiological function of polyamine acetylation is unknown, but the possibility of an effect on the association of polyamines with nucleic acids is discussed.     

Interconversion of putrescine,spermidine and spermine in goldfish and rat retina

Labelled putrescine is converted to spermidine and spermine in the retina of both the goldfish and of the rat, but the bulk remains as putrescine and spermidine in the goldfish retina whereas the bulk is present as spermine in the rat retina. Labelled spermidine is converted to spermine and to putrescine in the retina of both species, most remaining as spermidine in the goldfish retina whereas most is converted to spermine in the rat retina. Labelled spermine is converted to both spermidine and putrescine in the retina of both species with a greater conversion in the goldfish retina than in the rat retina. These results provide direct evidence of the interconversion of putrescine, spermidine and spermine in neural tissue from both fish and mammals and suggest that spermine should not be regarded solely as an end-product of putrescine metabolism but also as a source of spermidine and putrescine.The pattern of distribution of putrescine and the polyamines, spermidine and spermine, in goldfish retina is the reverse of that in rat retina: Putrescine is the most abundant in goldfish retina whereas spermine is most abundant in rat retina suggesting that the individual polyamines are of different importance in the two species.     

Binding and inhibition of human spermidine synthase by decarboxylated S-adenosylhomocysteine

Aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. Spermidine synthase (SpdS) is one of the most well‐studied enzymes in this biosynthetic pathway. The enzyme uses decarboxylated S‐adenosylmethionine and a short‐chain polyamine (putrescine) to make a medium‐chain polyamine (spermidine) and 5′‐deoxy‐5′‐methylthioadenosine as a byproduct. Here, we report a new spermidine synthase inhibitor, decarboxylated S‐adenosylhomocysteine (dcSAH). The inhibitor was synthesized, and dose‐dependent inhibition of human, Thermatoga maritima, and Plasmodium falciparum spermidine synthases, as well as functionally homologous human spermine synthase, was determined. The human SpdS/dcSAH complex structure was determined by X‐ray crystallography at 2.0 Å resolution and showed consistent active site positioning and coordination with previously known structures. Isothermal calorimetry binding assays confirmed inhibitor binding to human SpdS with K_d of 1.1 ± 0.3 μM in the absence of putrescine and 3.2 ± 0.1 μM in the presence of putrescine. These results indicate a potential for further inhibitor development based on the dcSAH scaffold.     

Aminooxy analogues of spermidine as inhibitors of spermine synthase and substrates of hepatic polyamine acetylating activity

Aminooxy analogues of spermidine, 1-aminooxy-3-N-[3-aminopropyl]- aminopropane (AP-APA) and N-[2-aminooxyethyl]-1,4-diaminobutane (AOE-PU), were tested as substrates or inhibitors of the enzymes involved in methionine and polyamine metabolism. Both compounds were good competitive inhibitors and poor substrates of spermine synthase, good substrates of cytosolic polyamine acetyltransferase, inactivators of S-adenosylmethionine decarboxylase and inhibitors of ornithine decarboxylase. AP-APA and AOE-PU showed K1-values of 1.5 and 186 microM as inhibitors of purified spermine synthase, and Km-values of 1.4 and 2.1 mM as substrates of the crude hepatic polyamine acetyltransferase activity. AP-APA was more potent than AOE-PU in crude enzyme preparations. Neither drug had any significant effect at 1 mM concentration on the activities of spermidine synthase, methionine adenosyltransferase, S-adenosylhomocysteine hydrolase, and methylthioadenosine phosphorylase. The results suggest that compounds of this type are valuable tools in unraveling the physiology of polyamines.     

Regulation of the activation of chloroplast fructose-1,6-bisphosphatase: inhibition by spermidine and spermine

The activation of chloroplast fructose-1,6-bisphosphatase by fructose-1,6-bisphosphate, Ca2+, DTT and chloroplast thioredoxin-f is prevented by either spermidine or spermine; on the contrary, other amino compounds do not replace polyamines in this reversible effect. On the other hand, neither spermidine nor spermine modify the catalysis of chloroplast fructose-1,6-bisphosphatase. The effect of spermidine, but not the effect of spermine, is reversed by increasing the concentration of Ca2+ in the activation; higher concentrations of Fructose-1,6-bisphosphate or thioredoxin-f do not restore the control activity. The present results suggest that other regulatory mechanisms may control the activation of fructose-1,6-bisphosphatase in chloroplasts.     

Reversal by aminoguanidine of the inhibition of proliferation of human fibroblasts by spermidine and spermine.

The inhibitory effect of the polyamines, spermidine and spermine, on the proliferation of human fibroblasts in culture was found to be reversed by the addition of aminoguanidine (AM), a specific and highly effective inhibitor of diamine oxidase (DAO) present in fetal calf serum (FCS). Aminoguanidine itself in concentration as high as 10(-3) M exhibited no effect upon cell proliferation nor did putrescine at similar concentrations. However, at higher concentrations of putrescine, cell proliferation was inhibited and this inhibition was unaffected by the addition of mM concentrations of AM. These studies support earlier hypotheses on the mechanisms of the toxic effects of polyamines on cell proliferation and establish further that the diamine oxidase-catalyzed metabolism of spermine and spermidine is necessary for their toxic effects in cell culture.     

Effects of spermidine synthase inhibition in cultured chick embryo fibroblasts.

The administration of bis-cyclohexylammonium sulfate (BCHS), a spermidine synthase inhibitor, to in vitro cultures of chick embryo fibroblasts caused a decrease in cellular spermidine levels and an increase in putrescine and spermine. Cell proliferation rate and DNA synthesis were also inhibited. As protein synthesis did not change, it would seem that low levels of cellular spermidine inhibit cell growth depressing DNA synthesis.     

Reversible inhibition of bacterial growth after specific inhibition of spermidine synthase by dicyclohexylamine.

The effect of dicyclohexylamine on seven freshly isolated bacterial strains of mastitis pathogens was studied. Streptococcus uberis was the most sensitive strain investigated, since 5 mM-dicyclohexylamine totally arrested its growth and 1.25 mM of the drug caused 60% growth inhibition. The Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains were also sensitive to the drug, but less so than Strep. uberis, since 5 mM drug caused only partial inhibition of growth. Micrococcus sp. and Klebsiella sp. grew in the presence of 10.0 mM-dicyclohexylamine, and, finally the growth of Streptococcus agalactiae was not at all affected by dicyclohexylamine. These different sensitivities towards dicyclohexylamine in vivo were paralleled by different sensitivities of the bacteria's spermidine synthase to the drug in vitro, and also by the ability of the drug to lower spermidine concentration in bacterial cells. Spermidine synthase from sensitive bacteria was inhibited by more than 90% by 50 microM-dicyclohexylamine in vitro, and the concentration of spermidine was decreased in E. coli and Ps. aeruginosa by 70% and in Strep. uberis by 95%, whereas in Strep. agalactiae 5 mM-dicyclohexylamine did not affect the concentration of spermidine at all. Dicyclohexylamine treatment led to the accumulation of putrescine in Strep. uberis. Spermidine synthesis catalysed by the extracts of Micrococcus sp. required 500 microM-dicyclohexylamine for 90% inhibition, and Strep. agalactiae contained a spermidine synthase that was still active at 1000 microM-dicyclohexylamine, The observed inhibition of growth was totally reversed by adding 50 microM-spermidine (final concentration) to the medium. Putrescine reversed the inhibition only when bacteria had a spermidine synthase activity insensitive to dicyclohexylamine. Spermine did not overcome the inhibition of growth caused by dicyclohexylamine, probably because it was not taken up by the bacterial cells used in this study. The inhibition of the growth by dicyclohexylamine (even in the case of Strep. uberis) was reversible in the sense that addition of 50 microM-spermidine 18 h after dicyclohexylamine still restored the growth rate of untreated controls.     

Hydroxylamine derivatives for regulation of spermine and spermidine metabolism

The biogenic polyamines spermine, spermidine, and their precursor putrescine are present in micro-to-millimolar concentrations in all cell types and are vitally important for their normal growth. High intracellular content of spermine and spermidine determines the multiplicity of the cellular functions of the polyamines. Many of these functions are not well characterized at the molecular level, ensuring the ongoing development of this field of biochemistry. Tumor cells have elevated polyamine level if compared with normal cells, and this greatly stimulates the search for new opportunities to deplete the intracellular pool of spermine and spermidine resulting in decrease in cell growth and even cell death. O-Substituted hydroxylamines occupy their own place among chemical regulators of the activity of the enzymes of polyamine metabolism. Varying the structure of the alkyl substituent made it possible to obtain within one class of chemical compounds highly effective inhibitors and regulators of the activity of all the enzymes of putrescine, spermine and spermidine metabolism (with the exception of FAD-dependent spermine oxidase and acetylpolyamine oxidase), effectors of the polyamine transport system, and even actively transported in cells “proinhibitor” of ornithine decarboxylase. Some principles for the design of specific inhibitors of these enzymes as well as the peculiarities of cellular effects of corresponding O-substituted hydroxylamines are discussed.     

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.     

Studies of the induction of spermidine/spermine N1-acetyltransferase using a specific antiserum

A specific antiserum to rat liver spermidine/spermine N1-acetyltransferase was used to study the induction of this protein. The antiserum had no effect on the spermidine acetylating capacity of crude nuclear extracts and very little effect on the activity present in crude cytosolic extracts from control rat tissues indicating that most of this activity is not due to spermidine/spermine N1-acetyltransferase. Treatment of rats with carbon tetrachloride, spermidine, thioacetamide, or methylglyoxal bis(guanylhydrazone) produced a substantial increase in the spermidine acetylating capacity of rat liver cytosolic extracts which was exclusively due to an increase in the immunoprecipitable spermidine/spermine N1-acetyltransferase protein. Exact measurement of the extent of this increase was not possible because the basal amount was too low to determine precisely but the amount of this enzyme increased about 250-fold with 6 h of treatment with carbon tetrachloride, about 25-fold at 6 h after spermidine, about 23-fold at 24 h after thioacetamide and up to 300-fold at 24 h after methylglyoxal bis(guanylhydrazone). Treatment of rats with spermidine also increased spermidine/spermine N1-acetyltransferase in other tissues including lung, kidney, and pancreas. The spermidine/spermine N1-acetyltransferase protein was found to turn over very rapidly with a half-life of about 15 min in thioacetamide-treated rats and 180 min after carbon tetrachloride.     

Polyamines spermidine and spermine as modulators of calcium-dependent immune processes

The polyamines spermidine and spermine are ubiquitous in animal cells and have been shown to bind to cell membranes. At least two types of immune processes, secretion of active substances during inflammation and lymphocyte activation in cellular immunity, involve cell membranes and calcium ions. Although these processes are activated by polyvalent substances, spermidine and spermine appear to inhibit them. This action of polyamines is discussed in the context of regulating both cell membrane fluidity and calcium fluxes.