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.     

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.     

Effect of inhibitors of S-adenosylmethionine decarboxylase on the contents of ornithine decarboxylase and S-adenosylmethionine decarboxylase in L1210 cells.

Treatment of L1210 cells with either of two inhibitors of S-adenosylmethionine decarboxylase (AdoMetDC), namely 5'-deoxy-5'-[N-methyl-N-[2-(amino-oxy)ethyl])aminoadenosine or 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)]aminoadenosine, produced a large increase in the amount of ornithine decarboxylase (ODC) protein. The increased enzyme content was due to a decreased rate of degradation of the protein and to an increased rate of synthesis, but there was no change in its mRNA content. The inhibitors led to a substantial decline in the amounts of intracellular spermidine and spermine, but to a big increase in the amount of putrescine. These results indicate that the content of ODC is negatively regulated by spermidine and spermine at the levels of protein translation and turnover, but that putrescine is much less effective in bringing about this repression. Addition of either spermidine or spermine to the cells treated with the AdoMetDC inhibitors led to a decrease in ODC activity, indicating that either polyamine can bring about this effect, but spermidine produced effects at concentrations similar to those found in the control cells and appears to be the physiologically important regulator. The content of AdoMetDC protein (measured by radioimmunoassay) was also increased by these inhibitors, and a small increase in its mRNA content was observed, but this was insufficient to account for the increase in protein. A substantial stabilization of AdoMetDC occurred in these cells, contributing to the increased enzyme content, but an increase in the rate of translation cannot be ruled out.     

Regulation of S-adenosylmethionine decarboxylase in L1210 leukemia cells. Studies using an irreversible inhibitor of the enzyme

A potent irreversible inhibitor of S-adenosylmethionine (AdoMet) decarboxylase, S-(5'-adenosyl)-methylthio-2-aminooxyethane (AdoMeSaoe), was used to study the regulatory control of this key enzyme in the polyamine biosynthetic pathway. Treatment of L1210 cells with the inhibitor completely eradicated the growth-induced rise in AdoMet decarboxylase activity, resulting in a marked decrease in cellular content of spermidine and spermine. The putrescine content, on the other hand, was greatly elevated. Although no detectable AdoMet decarboxylase activity was found in the L1210 cells after treatment with AdoMeSaoe, the cells contained 50-fold higher amounts of AdoMet decarboxylase protein, compared to untreated cells during exponential growth. Part of this increase was shown to be due to elevated synthesis of the enzyme. This stimulation appeared to be related to the decrease in cellular spermidine and spermine content, since addition of either one of the polyamines counteracted the rise in AdoMet decarboxylase synthesis. The synthesis rate was determined by immunoprecipitation of labeled enzyme after a short pulse with [35S]methionine. In addition to a protein that co-migrated with pure rat AdoMet decarboxylase (Mr approximately 32,000), the antibody precipitated a somewhat larger labeled protein (Mr approximately 37,000) that most likely represents the proenzyme form. Treatment of the L1210 cells with AdoMetSaoe also gave rise to a marked stabilization of the decarboxylase which contributed to the increase in its cellular protein content. Addition of spermidine did not significantly affect this stabilization, whereas the addition of spermine reduced the half-life of the enzyme to almost that of the control cells.     

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.     

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

Spermidine synthase (EC 2.5.1.16) was purified to apparent homogeneity (about 11 000-fold) from bovine brain by affinity chromatography, with S-adenosyl-(5')-3-thiopropylamine linked to Sepharose as the adsorbent. The enzyme preparation was free from S-adenosylmethionine decarboxylase (EC 4.1.1.50) and spermine synthase (EC 2.5.1.22) activities. The native enzyme had an apparent Mr of 70 000, was composed of two subunits of equal size, and had an isoelectric point at pH 5.22. The apparent Km values for putrescine and decarboxylated adenosylmethionine [S-adenosyl-(5')-3-methylthiopropylamine] were 40 microM and 0.3 microM respectively. Cadaverine and 1,6-diaminohexane could replace putrescine as the aminopropyl acceptor, although the reaction rates were only 6% and 1% respectively of that obtained with putrescine. Ethyl, propyl and carboxymethyl analogues of decarboxy-S-adenosylmethionine could act as propylamine donors. Both the reaction products, spermidine and 5'-methylthioadenosine, were mixed-type inhibitors of the enzyme. On the basis of initial-velocity and product-inhibition studies, a ping-pong reaction mechanism for the spermidine synthase reaction was ruled out.     

Effect of S-adenosyl-1,12-diamino-3-thio-9-azadodecane, a multisubstrate adduct inhibitor of spermine synthase, on polyamine metabolism in mammalian cells

The effects of the potent spermine synthase inhibitor S-adenosyl-1,12-diamino-3-thio-9-azadodecane (AdoDatad) on polyamine biosynthesis have been studied in transformed mouse fibroblasts (SV 3T3 cells) and in mouse leukemia cells (L1210). A dose-dependent decrease in intracellular spermine concentration was observed in both cell lines when grown in the presence of the inhibitor. A major difference in the effects seen in these two cell lines was the cytotoxicity observed in L1210 cells exposed to the inhibitor, which contrasted with little or no effects on growth of SV 3T3 cells treated similarly. Oxidative metabolism of the drug in L1210 cells was suggested by the fact that addition of aminoguanidine, an amine oxidase inhibitor, to the cell cultures ablated the cytotoxic effects of the inhibitor. Complete analysis of intracellular polyamines was carried out, together with analysis of S-adenosylmethionine, decarboxylated S-adenosylmethionine, and the inhibitor. These analyses revealed that, although the inhibitor had a dramatic effect on spermine biosynthesis in the cells studied, a compensatory increase in spermidine biosynthesis was observed. This resulted in no change in total polyamine concentrations in cells treated with inhibitors of either spermine synthase or spermidine synthase (Pegg et al., 1982) alone or in combination. In all cases, the concentration of the aminopropyl donor decarboxylated S-adenosylmethionine increased dramatically, thus allowing for the observed maintenance of total polyamine levels even in the presence of either one or both potent inhibitors of the aminopropyltransferases. Oxidative metabolism of the inhibitor complicates the interpretation of experiments carried out in the absence of amine oxidase inhibitors such as aminoguanidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of 1-aminooxy-3-aminopropane and S-(5'-deoxy-5'-adenosyl)methylthioethylhydroxylamine on ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase from Escherichia coli

1-Aminooxy-3-aminopropane (APA) was shown to be a potent competitive inhibitor (Ki = 1.0 nM) of partially purified Escherichia coli ornithine decarboxylase. APA did not inhibit S-adenosyl-L-methionine decarboxylase and spermidine from E. coli. S-(5'-Deoxy-5'-adenosyl)methylthioethylhydroxylamine (AMA), which is a structural analogue of decarboxylated S-adenosyl-L-methionine, was for the first time shown to be an irreversible inhibitor of bacterial S-adenosyl-L-methionine decarboxylase and a competitive inhibitor (Ki = 47 microM) of bacterial ornithine decarboxylase. AMA had no effect on spermidine synthase.     

Inhibition of translation of mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase by polyamines

The effect of spermidine and spermine on the translation of the mRNAs for ornithine decarboxylase and S-adenosylmethionine decarboxylase was studied using a reticulocyte lysate system and specific antisera to precipitate these proteins. It was found that the synthesis of these key enzymes in the biosynthesis of polyamines was much more strongly inhibited by the addition of polyamines than was either total protein synthesis or the synthesis of albumin. Translation of the mRNA for S-adenosylmethionine decarboxylase was maximal in a lysate which had been substantially freed from polyamines by gel filtration. Addition of 80 microM spermine had no significant effect on total protein synthesis and stimulated albumin synthesis but reduced the production of S-adenosylmethionine decarboxylase by 76%. Similarly, addition of 0.8 mM spermidine reduced the synthesis of S-adenosylmethionine decarboxylase by 82% while albumin and total protein synthesis were similar to that found in the gel-filtered lysate. Translation of ornithine decarboxylase mRNA was greater in the gel-filtered lysate than in the control lysate but synthesis of ornithine decarboxylase was stimulated slightly by low concentrations of polyamines and was maximal at 0.2 mM spermidine or 20 microM spermine. Higher concentrations were strongly inhibitory with a 70% reduction occurring at 0.8 mM spermidine or 150 microM spermine. Further experiments in which both polyamines were added together confirmed that the synthesis of ornithine and S-adenosylmethionine decarboxylases were much more sensitive to inhibition by polyamines than protein synthesis as a whole. These results indicate that an important part of the regulation of polyamine biosynthesis by polyamines is due to a direct inhibitory effect of the polyamines on the translation of mRNA for these biosynthetic enzymes.     

Inhibition of mammalian spermine synthase by N-alkylated-1,3-diaminopropane derivatives in vitro and in cultured rat hepatoma cells

A number of N-alkylated-1,3-diaminopropane derivatives [H2N-(CH2)3-NH-(CH2)nH, where n = 1-9] have been tested as potential inhibitors of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase. Among the compounds described in this paper, the most potent competitive inhibitor of spermine synthase, with respect to spermidine, is N-butyl-1,3-diaminopropane with Ki values of 11.9 nM and 10.4 nM for the HTC cell and bovine spleen enzymes respectively. Inhibition of spermine synthase by this alkylated amine is selective since spermidine synthase activity is not affected up to 100 microM N-butyl-1,3-diaminopropane at a range of 5-200 microM putrescine. Added to the culture medium of growing HTC cells, N-butyl-1,3-diaminopropane causes the expected changes in the polyamine levels with a marked decrease of spermine and an increase of spermidine. Under these conditions cell growth continues unabated. Such N-alkylated-1,3-diaminopropane derivatives may have considerable potential as tools for studying the role of polyamines and in particular the functions of spermine in cell multiplication and differentiation.     

S-adenosylmethionine decarboxylase from Leishmania donovani. Molecular, genetic, and biochemical characterization of null mutants and overproducers.

The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced from Leishmania donovani. The Delta adometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The Delta adometdc strains were incapable of growth in medium without polyamines; however, auxotrophy could be rescued by spermidine but not by putrescine, spermine, or methylthioadenosine. Incubation of Delta adometdc parasites in medium lacking polyamines resulted in a drastic increase of putrescine and glutathione levels with a concomitant decrease in the amounts of spermidine and the spermidine-containing thiol trypanothione. Parasites transfected with an episomal ADOMETDC construct were created in both wild type and Delta adometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the Delta adometdc L. donovani. In addition, ADOMETDC overproduction in wild type parasites alleviated the toxic effects of 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (MDL 73811), but not pentamidine, berenil, or methylglyoxyl bis(guanylhydrazone), all inhibitors of ADOMETDC activities in vitro. The molecular, biochemical, and genetic characterization of ADOMETDC establishes that it is essential in L. donovani promastigotes and a potential target for therapeutic validation.     

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.     

Spermidine biosynthesis as affected by osmotic stress in oat leaves

A new assay for the evaluation of spermidine (Spd) synthase activity was developed. It involves a coupled reaction and avoids the use of decarboxylated S-adenosylmethionine, which is unstable and not easily available. This assay was applied to assess changes in enzyme activity in oat leaves subjected to osmotic stress in the dark. The results indicate that osmotically-induced putrescine (Put) accumulation in cereals results not only from the activation of the arginine decarboxylase pathway, but also from the inhibition of the activity of Spd synthase, the enzyme which catalyzes the transformation of Put to Spd. Other possibilities which could contribute to the decline of Spd and spermine levels under osmotic stress are also discussed.Abbreviations ADC arginine decarboxylase - Dap diaminopropane - DFMA -difluoromethylarginine - MGBG methylglyoxal-bis-guanylhydrazone - MTA 5-deoxy-5-methylthioadenosine - ODC ornithine decarboxylase - PA polyamines - PAO polyamine oxidase - PCA perchloric acid - PLP pyridoxal phosphate - Put putrescine - SAM S-adenosylmethionine - dSAM decarboxylated S-adenosylmethionine - SAMDC S-adenosylmethionine decarboxylase - Spd spermidine - Spm spermine     

Polyamine-linked sepharoses: preparation and application to mammalian spermine synthase.

Seven different polyamine-linked Sepharose derivatives were prepared for the affinity chromatography of spermidine and spermine binding macromolecules: Spermine synthase from rat and hog brain was used as a model protein with a spermidine binding site. Comparative studies of the affinities of the enzymes for the seven matrixes suggested that two negative charges, three to four methylene groups apart, should be present at the decarboxylated S-adenosylmethionine binding site and should improve the binding of the enzyme to the Sepharose derivative. Two negative charges at the spermidine binding site would be expected to do the same. Three affinity matrixes linked with 1,17-diamino-4,9,14-triazaheptadecane, 1,21-diamino-4,9,13,18-tetraazaheneicosane, and 5-spermine carboxylic acid, respectively, had an affinity for spermine synthases higher than that of spermine-Sepharose, which has been used for the purification of spermine synthase. The first of these matrixes was used and proved to be effective for the purification.     

Effects of inhibitors of spermidine and spermine synthesis on polyamine concentrations and growth of transformed mouse fibroblasts

1. A number of compounds known to inhibit polyamine biosynthesis at various steps in the biosynthetic pathway were tested for their ability to inhibit growth and decrease polyamine concentrations in virally transformed mouse fibroblasts (SV-3T3 cells). 2. Virtually complete inhibition of growth was produced by the inhibitors of ornithine decarboxylase α-methylornithine and α-difluoromethylornithine and by the inhibitors of S-adenosylmethionine decarboxylase 1,1′-[(methylethanediylidene)dinitrilo]diguanidine and 1,1′-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine). The former inhibitors decreased putrescine and spermidine contents in the cells to very low values, whereas the latter substantially increased putrescine but decreased spermidine concentrations. The inhibitory effects of all of these inhibitors on cell growth could be prevented by the addition of spermidine, suggesting that spermidine depletion is the underlying cause of their inhibition of growth. 3. α-Difluoromethylornithine, which is an irreversible inhibitor of ornithine decarboxylase, was a more potent inhibitor of growth and polyamine production (depleting spermidine almost completely and spermine significantly) than α-methylornithine, which is a competitive inhibitor. This was not the case with the inhibitors of S-adenosylmethionine decarboxylase where 1,1′-[(methylethanediylidene)dinitrilo]diguanidine, a reversible inhibitor, was more active than 1,1′-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine), an irreversible inhibitor. It is suggested that this effect may be due to the lesser uptake and/or greater chemical reactivity of the latter compound. 4. Various nucleoside derivatives of S-adenosylhomocysteine that inhibited spermidine synthase in vitro did not have significant inhibitory action against polyamine accumulation in the cell. These compounds, which included S-adenosylhomocysteine sulphone, decarboxylated S-adenosylhomocysteine sulphone, decarboxylated S-adenosylhomocysteine sulphoxide and S-adenosyl-4-thio-butyric acid sulphone did not inhibit cell growth or polyamine content until cytotoxic concentrations were added. 5. 5′-Methylthioadenosine, 5′-isobutylthioadenosine and 5′-methylthiotubercidin, which inhibit aminopropyltransferase activity in vitro, all inhibited cell growth and decreased spermidine content. Although these compounds were most active against spermine synthase in vitro, they acted in the cell primarily to decrease spermidine content. Cell growth could not be restored to normal values by addition of spermidine, suggesting that these nucleosides have another inhibitory action towards cellular proliferation. 6. 5′-Methylthioadenosine and 5′-isobutylthioadenosine are degraded by a phosphorylase present in SV3T3 cells, yielding 5-methylthioribose-1-phosphate and 5-isobutylthioribose-1-phosphate respectively, and adenine. This degradation appears to decrease the inhibitory action towards cell growth, suggesting that the nucleosides themselves are exerting the inhibitory action. 5′-Methylthiotubercidin, which is not a substrate for the phosphorylase and is a competitive inhibitor of it, was the most active of these nucleosides in inhibiting cell growth and spermidine content. 5′-Methylthiotubercidin and α-difluoromethylornithine had additive effects on retarding cell growth, but not on cellular spermine accumulation, also suggesting that the primary growth-inhibiting action of the nucleoside was not on polyamine production. 7. These results support the concept that 5′-methylthioadenosine phosphorylase plays an important role in permitting cell growth to continue by preventing the build-up of inhibitory intracellular concentrations of 5′-methylthioadenosine.     

Inhibition of enzymes of polyamine biosynthesis by substrate-like O-substituted hydroxylamines

The biogenic amines spermine, spermidine, and putrescine are essential factors of cell growth and differentiation. To inhibit pyridoxal-5"-phosphate dependent ornithine decarboxylase and pyruvate dependent S-adenosylmethionine decarboxylase, key enzymes of polyamine biosynthesis, a system of substrate-like O-substituted hydroxylamines is suggested. The best of these compounds were active at nanomolar concentrations. High potency and specificity of this type of inhibitors are discussed in terms of structural similarity of E–I and E–S complexes.     

Regulation of S-adenosyl-L-methionine decarboxylase by 1-aminooxy-3-aminopropane: enzyme kinetics and effects on the enzyme activity in cultured cells

The kinetics of inactivation of adenosylmethionine decarboxylase of rat liver and of baby hamster kidney cells (BHK21/C31) by 1-aminooxy-3-aminopropane was studied. The apparent dissociation constants (Ki) for the hepatic and BHK21/C13 enzymes were 1.5 and 2.0 mM and the times of half-inactivation at infinite concentration of the inhibitor (tau 1/2) were 1.2 and 3.8 min, respectively. Treatment of BHK21/C13 with 0.5 mM 1-aminooxy-3-aminopropane prevented cell growth and depleted the cells of putrescine and spermidine within 1 day. The depletion of spermidine resulted in increased activity of S-adenosylmethionine decarboxylase which was due, at least partly, to the increase in the half-life of the enzyme activity. Because spermine levels were not significantly affected, it appears that spermidine is the principal feedback regulator of S-adenosylmethionine decarboxylase. So, 1-aminooxy-3-aminopropane is a very weak inhibitor of S-adenosylmethionine decarboxylase and the cellular effects can be correlated primarily with its inhibitory effects on ornithine decarboxylase and spermidine synthase. In cell-free systems, however, 1-aminooxy-3-aminopropane is likely to find use in unraveling the reaction mechanism of S-adenosylmethionine decarboxylase.     

S-adenosylmethionine decarboxylase degradation by the 26 S proteasome is accelerated by substrate-mediated transamination

The short-lived enzyme S-adenosylmethionine decarboxylase uses a covalently bound pyruvoyl cofactor to catalyze the formation of decarboxylated S-adenosylmethionine, which then donates an aminopropyl group for polyamine biosynthesis. Here we demonstrate that S-adenosylmethionine decarboxylase is ubiquitinated and degraded by the 26 S proteasome in vivo, a process that is accelerated by inactivation of S-adenosylmethionine decarboxylase by substrate-mediated transamination of its pyruvoyl cofactor. Proteasome inhibition in COS-7 cells prevents the degradation of S-adenosylmethionine decarboxylase antigen; however, even brief inhibition of the 26 S proteasome caused substantial losses of S-adenosylmethionine decarboxylase activity despite accumulation of S-adenosylmethionine decarboxylase antigen. Levels of the enzyme's substrate (S-adenosylmethionine) increased rapidly after 26 S proteasome inhibition, and this increase in substrate level is consistent with the observed loss of activity arising from an increased rate of inactivation by substrate-mediated transamination. Evidence is also presented that this substrate-mediated transamination accelerates normal degradation of S-adenosylmethionine decarboxylase, as the rate of degradation of the enzyme was increased in the presence of AbeAdo (5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine) (a substrate analogue that transaminates the enzyme); conversely, when the intracellular substrate level was reduced by methionine deprivation, the rate of degradation of the enzyme was decreased. Ubiquitination of S-adenosylmethionine decarboxylase is demonstrated by isolation of His-tagged AdoMetDC (S-adenosylmethionine decarboxylase) from COS-7 cells co-transfected with hemagglutinin-tagged ubiquitin and showing bands that were immunoreactive to both anti-AdoMetDC antibody and anti-hemagglutinin antibody. This is the first study to demonstrate that AdoMetDC is ubiquitinated and degraded by the 26 S proteasome, and substrate-mediated acceleration of degradation is a unique finding.     

Prolactin stimulation of Nb 2 node lymphoma cell division is inhibited by polyamine biosynthesis inhibitors

The mitogenic action of prolactin in Nb 2 node lymphoma cells was inhibited by two drugs which interfere with polyamine biosynthesis. At concentrations of 0.5 mM and above alpha-difluoromethyl ornithine (DFMO), which inhibits ornithine decarboxylase and the conversion of ornithine to putrescine, significantly attenuated the mitogenic effect of prolactin. This inhibition was prevented by the addition of putrescine, spermidine, or spermine to the culture medium. At concentrations of 1 microM and above methylglyoxal bis(guanylhydrazone) (MGBG), which inhibits S-adenosylmethionine decarboxylase and hence the conversion of putrescine to spermidine and spermine, abolished the mitogenic action of prolactin. This inhibition was prevented by the addition of spermidine or spermine, but not putrescine, to the culture medium. These studies show that ongoing polyamine biosynthesis is essential for prolactin to express its mitogenic effect in this lymphoma cell line.