Purification of rat liver S-adenosyl-L-methionine decarboxylase.
Ontology highlight
ABSTRACT:
SUBMITTER: Pegg AE
PROVIDER: S-EPMC1168114 | biostudies-other | 1974 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:S-Adenosyl-L-methionine decarboxylase (AdoMetDC) has been purified to near homogeneity from the Neff strain of Acanthamoeba castellanii. The holoenzyme molecular mass is 88.8 kDa, including two copies each of a 32.8 kDa alpha-subunit and a 10-15 kDa beta-subunit. The alpha-subunit contains the active site. It has an N-terminal pyruvoyl group, and the first 19 amino acids are 63 and 74% identical with comparable sequences from yeast and mammals, respectively. The apparent Km for S-adenosylmethionine (AdoMet) in the presence of 2 mM putrescine was 30.0 microM. The enzyme was stimulated 2-fold by putrescine, but was unaffected by spermidine. It was inhibited by the following anti-metabolites, listed with their Ki values: Berenil (0.17 microM), pentamidine (19.4 microM), propamidine (334 microM), hydroxystilbamidine (357 microM), methylglyoxal bis(guanylhydrazone) (604 microM) and ethidium bromide (1.3 mM). Activity of the enzyme fell to undetectable levels during cell differentiation (encystment).
| S-EPMC1134839 | biostudies-other
Project description:1. The activities of l-ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50) were dramatically enhanced in both the ventral prostate and the seminal vesicle of castrated rats in response to androgenic stimulation. The time course of the stimulation of ornithine decarboxylase together with the quantitatively different response of adenosylmethionine decarboxylase to testosterone treatment in the prostate gland and seminal vesicle indicated that the enhancement in polyamine synthesis in the ventral prostate may reflect both cellular proliferation and the restoration of the secretory functions of the organ. In the seminal vesicle, however, the stimulation of the polyamine-biosynthetic pathway more closely resembled the pattern found in other rat tissues, such as regenerating liver, undergoing compensatory growth. 2. Ornithine decarboxylase activity in the ventral prostate and especially in the seminal vesicle of sexually mature rat was diminished in vivo by various short-chain diamines such as 1,2-diaminoethane, 1,3-diaminopropane and putrescine (1,4-diaminobutane). These diamines had no direct effect on the enzyme activity in vitro. 3. In contrast with the marginal decrease in ornithine decarboxylase activity produced by diaminoethane in the ventral prostate of non-castrated animals, repeated injections of the latter amine completely prevented the intense stimulation of the enzyme activity in the ventral prostate and seminal vesicle of castrated rats at 24h after the commencement of testosterone treatment. 4. The decrease in ornithine decarboxylase activity observed after injections of diamines (putrescine) in the ventral prostate was apparently associated with a similar decrease in the amount of immunoreactive protein as revealed by immunotitration of the enzyme with antiserum to rat ornithine decarboxylase.
| S-EPMC1183783 | biostudies-other
Project description:The fate of S-adenosyl-L-methionine was studied in rat liver extracts by analysing the distribution of radioactivity from labelled adenosylmethionine in decomposition products, which were separated from each other by chromatographic and electrophoretic means. Marked non-enzymic degradation to adenine, pentosylmethionine, methylthioadenosine and homoserine was evident at pH 6.9-7.8. Enzymic cleavage to methylthioadenosine was stoichiometric with the accumulation of spermidine and could be totally prevented by inhibiting S-adenosyl-L-methionine decarboxylase. The results rule out the existence of adenosylmethionine cyclotransferase in rat liver and indicate that only two quantitatively significant enzymic processes are involved in hepatic adenosylmethionine degradation. Excluding nonenzymic decomposition, more than 99% of adenosylmethionine is demethylated and exclusively catabolized further by S-adenosyl-L-homocysteine hydrolase. Less than 1% of adenosylmethionine is decarboxylated and immediately utilized totally for polyamine biosynthesis.
| S-EPMC1144406 | biostudies-other
Project description:S-adenosyl-l-methionine decarboxylase (AdoMetDC) in the polyamine biosynthesis pathway has been identified as a suitable drug target in Plasmodium falciparum parasites, which causes the most lethal form of malaria. Derivatives of an irreversible inhibitor of this enzyme, 5'-{[(Z)-4-amino-2-butenyl]methylamino}-5'-deoxyadenosine (MDL73811), have been developed with improved pharmacokinetic profiles and activity against related parasites, Trypanosoma brucei. Here, these derivatives were assayed for inhibition of AdoMetDC from P. falciparum parasites and the methylated derivative, 8-methyl-5'-{[(Z)-4-aminobut-2-enyl]methylamino}-5'-deoxyadenosine (Genz-644131) was shown to be the most active. The in vitro efficacy of Genz-644131 was markedly increased by nanoencapsulation in immunoliposomes, which specifically targeted intraerythrocytic P. falciparum parasites.
| S-EPMC3940083 | biostudies-literature
Project description:1. Direct or indirect inhibitors of l-ornithine decarboxylase (EC 4.1.1.17), structurally related or unrelated to l-ornithine, including dl-alpha-difluoromethylornithine, alpha-methylornithine and 1,3-diaminopropane, used alone or in combination, decreased polyamine concentrations in rat hepatoma tissue culture (HTC) cells and increased S-adenosyl-l-methionine decarboxylase activity (EC 4.1.1.50). 2. Comparison of the catalytic properties of S-adenosyl-l-methionine from cells with elevated and normal activities revealed no apparent modification of the catalytic site as judged by affinity for the substrate, stimulation by di- and tri-amines and inhibition by methylglyoxal bis-(guanylhydrazone). 3. Actinomycin D and cycloheximide, and RNA and a proteinsynthesis inhibitor respectively, blocked the increase of S-adenosyl-l-methionine decarboxylase activity elicited by alpha-difluoromethylornithine. In polyamine-depleted cells the apparent half-life of elevated S-adenosyl-l-methionine decarboxylase activity, determined by inhibition of protein synthesis, was 2.5-fold longer than in control cells. The present results suggest that elevation of S-adenosyl-l-methionine decarboxylase activity by alpha-difluoromethylornithine is due to stabilization of the enzyme. 4. Restoration of the normal intracellular putrescine content, by addition of putrescine to the medium of polyamine-deficient cells, transiently increased S-adenosyl-l-methionine decarboxylase activity. Thereafter, intracellular conversion of putrescine into spermidine was accompanied by inactivation of the enzyme at a rate that was similar to that found on addition of spermidine itself. No relationship between total intracellular spermine content and S-adenosyl-l-methionine decarboxylase activity could be established. 5. Addition of 1mm-1,3-diaminopropane to polyamine-deficient cells did not cause a decrease in the activity of S-adenosyl-l-methionine decarboxylase, whereas addition of 1,5-diaminopentane (cadaverine) did. 1,3-Diamino-N-(3-aminopropyl)propane did not accumulate in cells treated with alpha-difluoromethylornithine and 1,3-diaminopropane, whereas addition of 1,5-diaminopentane led to the accumulation of 1,5-diamino-N-(3-aminopropyl)pentane. 1,3-Diamino-N-(3-aminopropyl)propane (10mum) was as effective as spermidine in decreasing S-adenosyl-l-methionine decarboxylase activity. Thus effectiveness of a diamine in decreasing enzyme activity is related to its capability of being converted into a closely structurally related homologue of spermidine by spermidine synthase. 6. The spermidine site of action appears to be post-translational since (a) the spermidine-induced decrease of S-adenosyl-l-methionine activity was not prevented by actinomycin D and (b) spermidine in the presence of cycloheximide led to a synergistic inactivation of the enzyme with a decay rate that progressively approached control values. Altogether these results are indirect evidence for a strict negative control of S-adenosyl-l-methionine decarboxylase by spermidine and substantiate previous findings [Mamont, Duchesne, Grove & Tardif (1978) Exp. Cell Res.115, 387-393]. Spermidine appears to act on some processes involved in denaturation and/or degradation of the enzyme protein. Putrescine appears to decrease the rate of these processes. The physiological significance of the regulatory control of S-adenosyl-l-methionine decarboxylase is discussed.
| S-EPMC1163012 | biostudies-other
Project description:BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe-4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe-4S](2+) cluster. Mutagenesis studies demonstrated that Cys31, Cys35, Cys38 in the C×××C×MC motif and Gly73, Gly74, Glu75, Pro76 in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met37 had little effect on its function. Our results indicate that BlsE represents a typical [4Fe-4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.
| S-EPMC3715490 | biostudies-literature
Project description:The putrescine-stimulated S-adenosyl-L-methionine decarboxylases from rat liver and yeast were strongly inhibited by Berenil and to a lesser extent by Pentamidine. Ten times greater drug concentrations were needed to achieve a similar level of inhibition of a Mg2+-stimulated bacterial enzyme. The inhibition was irreversible in that extensive dialyses or precipitation with (NH4)2SO4 did not restore enzyme activity. Putrescine did not protect the enzyme against Berenil, but adenosylmethionine either alone or with putrescine partially protected the irreversible action of Berenil. The compound 4,4'-diamidinodiphenylamine, which differs from Berenil only in lacking the azo group between benzene rings, was a weaker inhibitor than Berenil, and its inhibition was reversible. Berenil also inhibited the activity of adenosylmethionine decarboxylase in vivo, by depressing the activity of the enzyme in normal rat liver, for at least 24 h after a single injection (50 mg/kg body wt.) of the drug.
| S-EPMC1152717 | biostudies-other
Project description:Administration of methylglyoxal bis(guanylhydrazone) to leukaemic mice results in an early depression followed by a marked elevation of S-adenosyl-l-methionine decarboxylase activity. Further, there is an early prolonged increase in the activity of ornithine decarboxylase, the initial enzyme in the polyamine biosynthetic pathway. Because of the profound effects of methylglyoxal bis(guanylhydrazone) in vivo on the polyamine biosynthetic pathway, the drug can no longer be considered a specific inhibitor of spermidine synthesis.
| S-EPMC1166065 | biostudies-other
Project description:Polyamines (PAs) are nitrogenous molecules that are indispensable for cell viability and with an agreed-on role in the modulation of stress responses. Tobacco plants with downregulated SAMDC (AS-SAMDC) exhibit reduced PAs synthesis but normal levels of PA catabolism. We used AS-SAMDC to increase our understanding on the role of PAs in stress responses. Surprisingly, at control conditions AS-SAMDC plants showed increased biomass and altered developmental characteristics, such as increased height and leaf number. On the contrary, during salt stress AS-SAMDC plants showed reduced vigor when compared to the WT. During salt stress, the AS-SAMDC plants although showing compensatory readjustments of the antioxidant machinery and of photosynthetic apparatus, they failed to sustain their vigor. AS-SAMDC sensitivity was accompanied by inability to effectively control H2O2 levels and concentrations of monovalent and divalent cations. In accordance with these findings, we suggest that PAs may regulate the trade-off between growth and tolerance responses.
| S-EPMC4814703 | biostudies-literature
Project description:The present paper reports the tryptic digestion of N-ethylmaleimide-treated S-adenosyl-L-methionine synthetase (high- and low-Mr forms) and the isolation of the modified peptides by h.p.l.c. There is only one site modified after 5 min incubation, and the modification at this site correlates with the main activity decrease. The amino acid composition of this peptide was determined, and its localization in the sequence shows the modified residue as cysteine-150, which is located close to the putative ATP-binding site. Modification of the enzyme for 20 min led to the appearance of a second labelled peptide, which seems to be responsible for about a further 10% of the activity loss. The modification by N-ethylmaleimide of the enzyme was partially prevented in the presence of adenosine 5'-[beta gamma-imido]triphosphate and methionine, further supporting the hypothesis that the modified residues lie within the active site. Urea treatment of the enzyme, followed by modification with N-ethylmaleimide, produces the modification of 7 of the 10 cysteine residues present in the sequence. The results obtained were the same for either of the isoforms.
| S-EPMC1150192 | biostudies-other