S-adenosylmethionine decarboxylase from baker''s yeast.

1. S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1'[(methylethanediylidene)dinitrilo]diguanidine) [Pegg, (1974) Biochem J. 141, 581-583]. The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4. 2. S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity [5'-deoxyadenosyl-(5'),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16] during the purification procedure. 3. Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine. 4. Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine. The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine. 5. The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM. 6. Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme. 7. Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5'-phosphate.     

Effects of S-adenosyl-1,8-diamino-3-thiooctane on polyamine metabolism

Exposure of mammalian cells (transformed mouse fibroblasts or rat hepatoma cells) to S-adenosyl-1,8-diamino-3-thiooctane produced profound changes in the intracellular polyamine content. Putrescine was increased and spermidine was decreased, consistent with the inhibition of spermidine synthase by this compound, which is a potent and specific "transition-state analogue inhibitor" of the isolated enzyme in vitro. The spermine content of the cells was increased by exposure to this drug presumably since spermine synthase was able to use a greater proportion of the available decarboxylated S-adenosylmethionine when spermidine synthase was inhibited. The decarboxylated S-adenosylmethionine content rose substantially because the activity of S-adenosylmethionine decarboxylase was increased in response to the decline in spermidine. These results indicate that S-adenosyl-1,8-diamino-3-thiooctane is taken up by mammalian cells and is an effective inhibitor of spermidine synthase in vivo and that S-adenosylmethionine decarboxylase is regulated by the content of spermidine, but not of spermine. The growth of SV-3T3 cells was substantially reduced in the presence of S-adenosyl-1,8-diamino-3-thiooctane at concentrations of 50 microM or greater. Such inhibition was reversed by the addition of spermidine but not by putrescine. When SV-3T3 cells were exposed to 5 mM alpha-(difluoromethyl)ornithine and 50 microM S-adenosyl-1,8-diamino-3-thiooctane, the content of all polyamines was reduced. Putrescine and spermidine declined by more than 90% and spermine by 80%. Such cells grew very slowly unless spermidine was added.     

Polyamine synthesis in mammalian tissues. Isolation and characterization of spermine synthase from bovine brain

Spermine synthase, a propylamine transferase, which catalyses the biosynthesis of spermine from S-methyladenosylhomocystemine and spermidine has been purified to an apparent homogeneity (about 6000-fold) from bovine brain using spermine-Sepharose affinity chromatography. The enzyme preparation was free from S-adenosylmethionine decarboxylase and spermidine synthase activities. The molecular Stokes radius of the enzyme was calculated to be 4.16 nm. The enzyme has an apparent molecular weight of approximately 88 000, composing of two subunits of equal size. The enzyme showed a broad pH optimum between 7.0 and 8.0 and an acidic isoelectric point at pH 5.10. The apparent Km values for S-methyladenosylhomocysteamine was 0.6 microM and about 60 microM for spermidine. The enzyme showed strict specificity to spermidine as the propylamine acceptor. Both the reaction products, spermine and 5'-methylthioadenosine inhibited the enzyme activity, methylthioadenosine being a powerful competitive inhibitor with respect to S-methyladenosylhomocysteamine (Ki value of about 0.3 microM). Putrescine also inhibited competitively with respect to spermidine (Ki value of about 1.7 mM). Spermine synthase had no requirements for metal or other cofactors.     

Effects of certain 5'-substituted adenosines on polyamine synthesis: selective inhibitors of spermine synthase

A number of nucleosides related to S-adenosylmethionine were tested for their inhibitory action on three enzymes involved in the biosynthesis of polyamines. The particular objective of the experiments was to determine whether any of the compounds could be used as selective inhibitors of the synthesis of spermine by spermine synthase. None of the nucleosides examined were potent inhibitors of S-adenosylmethionine decarboxylase. 5'-[(3-Aminopropyl)amino]-5'-deoxyadenosine dihydrochloride was quite a strong inhibitor of spermidine synthase (I50 of 7 microM) but was more than an order of magnitude less active than S-adenosyl-1,8-diamino-3-thiooctane, which is a mechanism-based inhibitor of this enzyme. 5'-[(3-Aminopropyl)amino]-5'-deoxyadenosine also inhibited spermine synthase with an I50 of 17 microM, but more selective inhibition of spermine synthase was produced by 9-[6(RS),8-diamino-5,6,7,8-tetradeoxy-beta-D-ribo-octofuranosyl]-9 H-purin-6- amine (I50 of 12 microM) and by dimethyl(5'-adenosyl)sulfonium perchlorate (I50 of 8 microM) since these compounds were much less active against spermidine synthase. Both 9-[6(RS),8-diamino-5,6,7,8-tetradeoxy-beta-D-ribo-octofuranosyl]-9 H-purin-6- amine and dimethyl(5'-adenosyl)sulfonium perchlorate were able to reduce the synthesis of spermine in SV-3T3 cells, but there was a compensatory increase in the concentration of spermidine, and there was no effect on cell growth. These results and those from experiments in which these spermine synthesis inhibitors were combined with inhibitors of spermidine synthase and ornithine decarboxylase indicated that the cells compensated for the inhibition of the aminopropyltransferases by increasing the production of decarboxylated S-adenosylmethionine and putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversal of the deoxyhypusine synthesis reaction. Generation of spermidine or homospermidine from deoxyhypusine by deoxyhypusine synthase

Deoxyhypusine synthase catalyzes the first step in hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) synthesis in a single cellular protein, eIF5A precursor. The synthesis of deoxyhypusine catalyzed by this enzyme involves transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein to form a deoxyhypusine-containing eIF5A intermediate, eIF5A(Dhp). We recently discovered the efficient reversal of deoxyhypusine synthesis. When eIF5A([3H]Dhp), radiolabeled in the 4-aminobutyl portion of its deoxyhypusine residue, was incubated with human deoxyhypusine synthase, NAD, and 1,3-diaminopropane, [3H]spermidine was formed by a rapid transfer of the radiolabeled 4-aminobutyl side chain of the [3H]deoxyhypusine residue to 1,3-diaminopropane. No reversal was observed with [3H]hypusine protein, suggesting that hydroxylation at the 4-aminobutyl side chain of the deoxyhypusine residue prevents deoxyhypusine synthase-mediated reversal of the modification. Purified human deoxyhypusine synthase also exhibited homospermidine synthesis activity when incubated with spermidine, NAD, and putrescine. Thus it was found that [14C]putrescine can replace eIF5A precursor protein as an acceptor of the 4-aminobutyl moiety of spermidine to form radiolabeled homospermidine. The Km value for putrescine (1.12 mM) as a 4-aminobutyl acceptor, however, is much higher than that for eIF5A precursor (1.5 microM). Using [14C]putrescine as an acceptor, various spermidine analogs were evaluated as donor substrates for human deoxyhypusine synthase. Comparison of spermidine analogs as inhibitors of deoxyhypusine synthesis, as donor substrates for synthesis of deoxyhypusine (or its analog), and for synthesis of homospermidine (or its analog) provides new insights into the intricate specificity of this enzyme and versatility of the deoxyhypusine synthase reaction.     

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.     

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.     

Biosynthesis of spermidine, a direct precursor of pyrrolizidine alkaloids in root cultures of Senecio vulgaris L.

 The polyamine spermidine is an essential biosynthetic precursor of pyrrolizidine alkaloids. It provides its aminobutyl group which is transferred to putrescine yielding homospermidine, the specific building block of the necine base moiety of pyrrolizidine alkaloids. The enzymatic formation of spermidine was studied in relation to the unique role of this polyamine as an alkaloid precursor. S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and spermidine synthase (SPDS, EC 2.5.1.16) from root cultures of Senecio vulgaris were partially purified and characterized. The SAMDC-catalyzed reaction showed a pH optimum of 7.5, that of SPDS an optimum of 7.7. The K _m value of SAMDC for its substrate S-adenosylmethionine (SAM) was 15 μM, while the apparent K _m values of SPDS for its substrates decarboxylated SAM (dSAM) and putrescine were 4 μM and 21 μM, respectively. The relative molecular masses of the two enzymes, determined by gel filtration, were 29 000 (SAMDC) and 37 000 (SPDS). Studies with various potential inhibitors revealed, for most inhibitors, profiles that were similar to those established with the respective enzymes from other plant sources. However, putrescine which is not known to be an inhibitor of plant SAMDC, strongly inhibited the enzyme from S. vulgaris roots. Spermidine synthase was sensitive to inhibition by its product spermidine. In the presence of the stationary tissue concentrations of the two polyamines (ca. 0.1 mM each) the activities of SAMDC and SPDS would be inhibited by >80%. The results are discussed in relation to the role of spermidine in primary and secondary metabolism of alkaloid-producing S. vulgaris root cultures. Received: 15 September 1999 / Accepted 10 December 1999     

Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes

Spermidine synthase (EC 2.5.1.16) was purified to homogeneity for the cytosol of soybean (Glycine max) axes using ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, omega-aminooctyl-Sepharose and ATPA-Sepharose. The molecular mass of the enzyme estimated by gel filtration and SDS-PAGE is 74 kDa. Cadaverin and 1,6-diaminohexane could not replace putrescine as the aminopropyl acceptor. Kinetic behaviors of the substrate are consistent with a ping pong mechanism. The kinetic mechanism is further supported by direct evidence confirming the presence of an aminopropylated enzyme and identification of product, 5'-deoxy-5'-methylthioadenosine, prior to adding putrescine. The Km values for decarboxylated S-adenosylmethionine and putrescine are 0.43 microM and 32.45 microM, respectively. Optimum pH and temperature for the enzyme reaction are 8.5 and 37 degrees C, respectively. The enzyme activity is inhibited by N-ethylmaleimide and DTNB, but stimulated by Co2+, Cu2+ and Ca2+ significantly, suggesting that these metal ions could be the cellular regulators in polyamine biosynthesis.     

Purification of spermidine synthase from rat ventral prostate by affinity chromatography on immobilized S-adenosyl(5′)-3-thiopropylamine

A novel affinity chromatographic adsorbent was developed for purification of spermidine synthase from rat prostate. The adsorbent (S-adenosyl(5′)-3-thiopropylamine-Sepharose) possesses a ligand structurally similar to S-adenosyl(5′)-3-methylthiopropylamine (decarboxy AdoMet), a substrate of spermidine synthase. The S-adenosyl(5′)-3-thiopropylamine-Sepharose was prepared by an alkylation on sulfur of S-adenosyl-3-thiopropylamine by bromoacetamidohexyl-Sepharose under mild acidic conditions. The enzyme has been purified to homogeneity in 40% yield by using DEAE-cellulose, affinity chromatography employing S-adenosyl(5′)-3-thiopropylamine-Sepharose, and gel filtration. The enzyme had a molecular weight of approximately 73,000 and was composed of two subunits of equal size. The specificity of the reaction was rather strict, but cadaverine could replace putrescine as the aminopropyl acceptor, and the rate was 1/20th of the rate for spermidine formation. Apparent K_m values for putrescine and decarboxy AdoMet were 0.1 mm and 1.1 μm, respectively. Inhibition by decarboxy AdoMet and 5′-deoxy-5′-methylthioadenosine was observed. The inhibition by 5′-deoxy-5′-methylthioadenosine was partially noncompetitive with respect to decarboxy AdoMet.     

Effects of S-adenosyl-1,8-diamino-3-thio-octane and S-methyl-5''-methylthioadenosine on polyamine synthesis in Ehrlich ascites-tumour cells.

The rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are negatively regulated by the polyamines spermidine and spermine. In the present work the spermidine synthase inhibitor S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and the spermine synthase inhibitor S-methyl-5'-methylthioadenosine (MMTA) were used to evaluate the regulatory role of the individual polyamines. Treatment of Ehrlich ascites-tumour cells with AdoDATO caused a marked decrease in spermidine content together with an accumulation of putrescine and spermine. Treatment with MMTA, on the other hand, gave rise to a marked decrease in spermine, with a simultaneous accumulation of spermidine. A dramatic increase in the activity of AdoMetDC, but not of ODC, was observed in MMTA-treated cells. This increase appears to be unrelated to the decrease in spermine content, because a similar rise in AdoMetDC activity was obtained when AdoDATO was given in addition to MMTA, in which case the spermine content remained largely unchanged. Instead, we show that the increase in AdoMetDC activity is mainly due to stabilization of the enzyme, probably by binding of MMTA. Treatment with AdoDATO had no effects on the activities of ODC and AdoMetDC, even though it caused a precipitous decrease in spermidine content. The expected decrease in spermidine-mediated suppression of ODC and AdoMetDC was most probably counteracted by the simultaneous increase in spermine. The combination of AdoDATO and MMTA caused a transient rise in ODC activity. Concomitant with this rise, the putrescine and spermidine contents increased, whereas that of spermine remained virtually unchanged. The increase in ODC activity was due to increased synthesis of the enzyme. There were no major effects on the amount of AdoMetDC mRNA by treatment with the inhibitors, alone or in combination. However, the synthesis of AdoMetDC was slightly stimulated in cells treated with MMTA or AdoDATO plus MMTA. The present study demonstrates that regulation of neither ODC nor AdoMetDC is a direct function of the polyamine structure. Instead, it appears that the biosynthesis of the polyamines is feedback-regulated by the various polyamines at many different levels.     

Human deoxythymidine kinase. I. Purification and general properties of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia.

Two forms of deoxythymidine kinase from blast cells of acute myelocytic leukemia were identified by electrophoresis. One was associated mainly with the cytoplasm and the other with mitochondria. Both isozymes were separated and purified by differential affinity column chromatography which resulted in 2416- and 1634-fold purification of the cytoplasmic and mitochondrial enzymes, respectively. Affinity gel was prepared by linkage through position 3' of deoxythymidine. Each enzyme had the same electrophoretic mobility in the purified state as it did in the enzyme derived from the corresponding subcellular fraction of the homogenate. Thymidine phosphorylase was not retarded by the affinity column. The purified cytoplasmic and mitochondrial deoxythymidine kinase had different molecular weights, sensitivities to inhibition by ammonium sulfate, activation energies for the reaction and divalent cation requirements. Adenosine, guanosine, and cytosine 3':5'-monophosphates, putrescine, spermine, and spermidine were neither activators nor inhibitors of either deoxythymidine kinase.     

Deoxyhypusine synthase from tobacco. cDNA isolation, characterization, and bacterial expression of an enzyme with extended substrate specificity.

Deoxyhypusine synthase catalyzes the formation of a deoxyhypusine residue in the translation eukaryotic initiation factor 5A (eIF5A) precursor protein by transferring an aminobutyl moiety from spermidine onto a conserved lysine residue within the eIF5A polypeptide chain. This reaction commences the activation of the initiation factor in fungi and vertebrates. A mechanistically identical reaction is known in the biosynthetic pathway leading to pyrrolizidine alkaloids in plants. Deoxyhypusine synthase from tobacco was cloned and expressed in active form in Escherichia coli. It catalyzes the formation of a deoxyhypusine residue in the tobacco eIF5A substrate as shown by gas chromatography coupled with a mass spectrometer. The enzyme also accepts free putrescine as the aminobutyl acceptor, instead of lysine bound in the eIF5A polypeptide chain, yielding homospermidine. Conversely, it accepts homospermidine instead of spermidine as the aminobutyl donor, whereby the reactions with putrescine and homospermidine proceed at the same rate as those involving the authentic substrates. The conversion of deoxyhypusine synthase-catalyzed eIF5A deoxyhypusinylation pinpoints a function for spermidine in plant metabolism. Furthermore, and quite unexpectedly, the substrate spectrum of deoxyhypusine synthase hints at a biochemical basis behind the sparse and skew occurrence of both homospermidine and its pyrrolizidine derivatives across distantly related plant taxa.     

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.     

Influence of polyamines on the activity of DNA polymerase I from Escherichia coli.

The influence of polyamines on the various activities of DNA polymerase I from Escherichia coli (EC 2.7.7.7) has been investigated. For all high molecular weight DNAs spermine and spermidine caused up to 80% inhibition when present in high concentrations, i.e. above 1 mM for spermine and 2 mM for spermidine. In the presence of low concentrations of polyamines a small activation was seen for some DNAs. The diamines cadaverine and putrescine had little influence on the rate of synthesis with natural occurring DNAs. In the case of d(A--T)n the activation/inhibition was found to be markedly dependent on the molecular weight of the samples used. With a low molecular weight DNA, 5.6 S, addition of spermidine resulted in up to 3-fold stimulation of activity. The activation was dependent on the concentration of MgCl2 and ionic strength; increasing concentration of these gave a decrease in the degree of activation. Polyamines also had a dramatic effect on the rate of synthesis using the homopolymers (dA)n . (dT)10 and (rA)n . (dT)10 . (20:1) as primers. Putrescine, in particular, increased the activity up to 10-fold with (rA)n . (dT)10 and somewhat less for (dA)n . (dT)10. The apparent Km for the primer (rA)n . (dT)10 decreased approx. 35-fold in the presence of 6.6 mM putrescine. There was no influence on the apparent Km for dTTP. The influence of polyamines on both the 5' leads to 3' and 3' leads to 5' nuclease activity was also investigated. Inhibition of nuclease activity was observed in the presence of polyamines, particularly with spermine. Thus with d(A--T)n and T7 DNA as substrates addition of 0.7 mM spermine resulted in almost complete inhibition of the activity. The dramatic inhibition observed with high concentrations of spermine (spermidine) both in the case of polymerizing and nuclease activity is thought to be due to polyamine-induced aggregation of DNA molecules.     

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. Mg2+ 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 Km-value for adenosylmethionine was determined to be 17 microM. Methylglyoxal bis(guanylhydrazone) and berenil competitively inhibited the enzyme activity; the apparent Ki-values were found to be 0.24 microM and 0.11 microM, 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.     

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.