S-adenosylmethionine Analog Research Articles

Effects of selenomethionine on cell growth and on S-adenosylmethionine metabolism in cultured malignant cells

The effects of selenomethionine (SeMet) on the growth of 17 cultured cell lines were studied. SeMet in the culture medium of three hepatoma cell lines promoted cell growth at subcytotoxic levels (1-20 microM), but the growth of malignant lymphoid and myeloid cells was not stimulated. L-SeMet was cytotoxic to all 17 cell lines when assayed after culture for 3-10 days. A 50% growth inhibition was observed by 30-160 microM-SeMet in a culture medium containing 100 microM-methionine. SeMet cytotoxicity to normal (fibroblasts) and malignant cells was rather similar, excluding specific antineoplastic cytotoxicity. Cytotoxicity was increased by decreasing concentrations of methionine. The DL form of SeMet was less cytotoxic than the L form. L-SeMet was metabolized to a selenium analogue of S-adenosylmethionine approximately as effectively as the natural sulphur analogue methionine in malignant R1.1 lymphoblasts. Concomitantly, S-adenosylmethionine pools were decreased. This occurred early and at cytotoxic SeMet levels. Methionine adenosyltransferase activity was not altered by SeMet treatment. ATP pools were not affected early, and decreases in the synthesis of DNA and protein took place late and were apparently related to cell death. RNA synthesis was slightly stimulated at low cytotoxic SeMet levels by 24 h, but was markedly inhibited after 48 h. The SeMet analogue of S-adenosylmethionine could be effectively utilized in a specific enzymic transmethylation. Neither S-adenosylhomocysteine nor its selenium analogue accumulated in the treated cells. These findings together suggest a direct or indirect involvement of S-adenosylmethionine metabolism in SeMet cytotoxicity, but exclude a gross blockage of transmethylations.

Effect of Analogues of S-Adenosylmethionine on in vitro Polyadenylation by Vesicular Stomatitis Virus

Other workers have reported that vesicular stomatitis virus makes aberrantly long polyadenylic acid [poly(A)] tracts in the presence of S-adenosylhomocysteine (S-Ado-Hcy). In the work reported in this paper, the effects of various analogues of S-adenosylmethionine (S-Ado-Met) and ATP on polyadenylation in an in vitro transcription system were examined to determine whether S-Ado-Hcy exerted its effect on polyadenylation due to its relationship to S-Ado-Met or to ATP. It appeared that compounds which affected polyadenylation were those which were closely related to S-Ado-Met and that had the same L-aminoacyl side chain [(COOH)-CH(NH)2-CH2-CH2-]; the nature of the substituent at the -S+(CH3)- position of S-Ado-Met was less important. These analogues appeared to compete with S-Ado-Met for a binding site(s). These data support a model whereby compounds binding at an S-Ado-Met-binding site may have allosteric effects by causing or preventing conformational changes which are involved in polyadenylation reactions, perhaps by affecting the rate of polyadenylation or of termination.

Guanine nucleotide-dependent carboxyl methylation of mammalian membrane proteins.

A guanine nucleotide-dependent protein carboxyl methylation is demonstrated in mammalian cell membranes. The methylation of membrane proteins of Mr 20,000-23,000 requires S-adenosylmethionine, GTP or nonhydrolyzable GTP analogs, and a cytoplasmic methyltransferase. The protein methyl groups are stable at neutral pH and under basic conditions hydrolyze to produce methanol. The specific methyl acceptor proteins and methyltransferases varied between tissues and cell types, suggesting that these methylations have cell-specific functions. The guanine nucleotide-dependent carboxyl methylations provide a possible mechanism for regulating the function of GTP-binding membrane proteins in the transduction of receptor-mediated signals of eukaryotic cells.

General synthetic approach to stable nitrogen analogs of S-adenosylmethionine

General synthetic approach to stable nitrogen analogs of S-adenosylmethionine

Effect of inhibitors of S-adenosylmethionine decarboxylase on polyamine content and growth of L1210 cells.

Analogues of S-adenosylmethionine that were designed as inhibitors of S-adenosylmethionine decarboxylase were tested for their abilities to inhibit the purified enzyme from rat prostate. The most potent inhibitors were 5'-deoxy-5'-[N-methyl-N-[2-(aminooxy)ethyl]amino]adenosine (MAOEA) and 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)amino]adenosine (MHZPA), which had I50 values of 400 nM and 70 nM, respectively, when added directly to the assay medium under standard conditions. These compounds were irreversible inactivators of the enzyme, and more than 95% of the activity was lost within 15 min of exposure to 5 microM MAOEA or 0.5 microM MHZPA. Both inhibitors led to a large reduction in the content of decarboxylated S-adenosylmethionine in L1210 cells and to a substantial decrease in the production of 5'-(methylthio)adenosine by these cells. These results are consistent with their bringing about an inhibition of S-adenosylmethionine decarboxylase activity in the cell which leads to a reduction in the synthesis of spermidine and spermine. Analysis of the polyamine content in L1210 cells exposed to 100 microM MAOEA or 50 microM MHZPA showed that this was the case and that putrescine levels were greatly increased while spermidine and spermine content declined. The combined application of 100 microM MAOEA and 5 mM alpha-(difluoromethyl)ornithine (an ornithine decarboxylase inhibitor) to L1210 cells completely prevented the synthesis of putrescine, spermidine, and spermine for up to 48 h. The reduction in polyamine content brought about by MHZPA or MAOEA could be partially prevented by the addition of decarboxylated S-adenosylmethionine to the culture medium. These inhibitors also brought about an inhibition of cell growth which could be reversed by the addition of spermidine.(ABSTRACT TRUNCATED AT 250 WORDS)

New Substrate Analogues as Inhibitors of S-Adenosylmetfflonine Decarboxylase

Abstract Analogues of S-adenosylmethionine with modifications in the 5′-group were prepared as potential inhibitors of S-adenosylmethionine decarboxylase (AdoMet-DC). These new analogues contained carbonyl-reactive end groups in the 5′-side chain, designed to interact favorably with the pyruvate prosthetic group of AdoMet-DC. Several of the analogues proved to be outstanding inhibitors of the enzyme. The analogues were also evaluated for their activity against human cytomegalovirus in vitro.

Inhibition of the methionine cycle enzymes

Inhibition of the enzymes involved in the production of 1-aminocyclopropane-1-carboxylic acid (ACC) and the subsequent salvage of methionine from 5′-methylthioadenosine (MTA) was studied. Possible product inhibition of ACC synthase, which converts S-adenosylmethionine (SAM) to ACC and MTA, and MTA nucleosidase, which hydrolyses MTA to 5-methylthioribose (MTR) and adenine, was investigated. ACC synthase was weakly inhibited by MTA ( K i = 0.2mM). MTA nucleosidase was inhibited by adenine competitively ( K i = 40μM), but not by MTR. Some analogues of the enzymes' substrates were inhibitory. ACC synthase was strongly and competitively inhibited by sinefungin, a SAM analogue ( K i = 2μM); MTA nucleosidase was inhibited by various MTA analogues, including 5′-chloroformycin, 5′-chloroadenosine, and 5′-ethylthioadenosine. The conversion of MTR to methionine in avocado extract was inhibited by the MTR analogues 5-chlororibose and 5-ethylthioribose, which exert their inhibitory effects by inhibiting MTR kinase. The capacity to convert MTR to methionine in ripening apple tissue appears to be ample; thus, this conversion does not appear to be a limiting factor of ethylene production.

Cell cycle effects in yeast cells of the antifungal agent Sinefungin

The S-adenosylmethionine (AdoMet) analog Sinefungin (SF) caused actively dividing cells of the yeast Saccharomyces cerevisiae to arrest within one cell cycle as unbudded cells. Reciprocal shift experiments showed that these cells were blocked in performance of the cell cycle regulatory step “start”. Both protein and RNA synthesis rates were only moderately affected during SF-mediated cell cycle arrest; these results indicate that SF provokes a different sort of metabolic response than found upon treatment with other “start”-arrest compounds.

Cell cycle effects in yeast cells of the antifungal agent Sinefungin

The S-adenosylmethionine (AdoMet) analog Sinefungin (SF) caused actively dividing cells of the yeast Saccharomyces cerevisiae to arrest within one cell cycle as unbudded cells. Reciprocal shift experiments showed that these cells were blocked in performance of the cell cycle regulatory step “start”. Both protein and RNA synthesis rates were only moderately affected during SF-mediated cell cycle arrest; these results indicate that SF provokes a different sort of metabolic response than found upon treatment with other “start”-arrest compounds.

Antifungal antibiotics and Siba inhibit 1-aminocyclopropane-1-carboxylic acid synthase activity

The antifungal antibiotics Sinefungin and A9145C isolated from Streptomyces griseolus and the synthetic nucleoside Siba, which are analogs of S-adenosylmethionine, inhibit the activity of 1-aminocyclopropane 1-carboxylic acid synthase from tomato fruits. Sinefungin and Siba were shown to be more potent inhibitors than A9145C. In extracts of green fruits, the enzyme activity was inhibited by Sinefungin with an I 50 of 1 μM, which was similar to that caused by aminoethoxyvinylglycine, and by Siba with an I 50 of 100 μM; in extracts from red tomatoes, the I 50's were 25 μM and 100 μM, respectively. The inhibition of ACC synthase by these analogs could be reversed by gel filtration chromatography.

The synthesis of the N-methyl analogue of S-adenosylmethionine; N.M.R. observation of diastereomers

The N-methyl analogue (1; X = N-Me) of S-adenosylmethionine has been synthesized; the intermediates and the end-product are racemic at the amino acid α-carbon, and show diastereomeric splitting in their 13C n.m.r. spectra.

Reversed-phase ion-pair liquid chromatographic procedure for the simultaneous analysis of S-adenosylmethionine, its metabolites and the natural polyamines

A method using reversed-phase ion-pair liquid chromatography with dual detection was developed for the simultaneous determination of the S-adenosylmethionine (SAM) analogues and the natural polyamines. The separation is obtained with a gradient elution and by adjusting the concentration of octanesulfonic acid used as ion-pairing agent, the ionic strength of the eluent, the pH and the acetonitrile content of the eluents. The SAM analogues are analyzed by UV detection at 254 nm and the polyamines by fluorescence detection after post-column derivatization with o-phthalaldehyde. The method allows the determination of the SAM analogues and the polyamines in one single run by direct injection of tissue extracts. The procedure is applied to the study in rats and in hepatoma tissue culture cells of the biochemical effects of α-difluoromethylornithine, a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase.

Induction of hemoglobin synthesis by xylosyladenine in murine erythroleukemia cells. Metabolism of xylosyladenine and effects on transmethylation.

The adenosine analog xylosyladenine is a potent inducer of hemoglobin synthesis in Friend virus-infected murine erythroleukemia (MEL) cells. In cultures treated with 0.1 microM xylosyladenine and an inhibitor of adenosine deaminase, 80% of the cells accumulated hemoglobin. Under these conditions, cell growth was inhibited by 50%. No effect was observed in the absence of adenosine deaminase inhibition. An adenosine kinase-deficient MEL subline was isolated and was found to be resistant to induction by xylosyladenine. Treated cells accumulated substantial amounts of the xylofuranosyl analogs of ATP, S-adenosylmethionine, and S-adenosylhomocysteine, indicating that metabolites of xylosyladenine participate in S-adenosylmethionine-mediated transmethylation reactions. Measurements of in vivo nucleic acid methylation showed that xylosyladenine causes a marked inhibition of 2'-O-methyluridine, 2'-O-methylcytidine, 5-methyluridine, and 5-methylcytidine formation in the RNA of treated cells. DNA methylation was not inhibited. These data suggest that the xylofuranosyl analogs of S-adenosylmethionine and/or S-adenosylhomocysteine can inhibit intracellular RNA methylation in MEL cells while having little or no such effect on DNA methylation.

Methylation of chemotaxis-specific proteins in Escherichia coli cells permeable to S-adenosylmethionine.

Using a modification of the EGTA treatment of Oishi and Smith [Oishi, M., & Smith, C. L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3569], Escherichia coli cells have been made permeable to S-adenosylmethionine and other related molecules in order to facilitate the study of methylation in chemotaxis. The permeable cells are nonmotile but respond to chemotactic stimuli by reversible methylation of their methyl-accepting chemotactic proteins (MCP I and MCP II) in a manner similar to that of untreated, motile cells. Addition of S-adenosyl-L-[methyl-3H]methionine to the permeable cells specifically labels two proteins, MCP I and MCP II. Methylation of these MCP's is dependent on the presence of wild-type gene products of flaI, flaA, cheB, cheX, tsr, and tar. The extent of methylation of the MCP's is affected by the presence of attractants or repellents: addition of attractant increases the steady-state level of methylation; addition of repellent causes rapid demethylation to a new steady-state level. Methylation is inhibited by the addition of the transmethylase inhibitors A9145C and Sinefungin, which are S-adenosylmethionine analogues, and by S-adenosylhomocysteine.

5-methylcytosine formation in wheat embryo DNA

Summary The methylation of cytosine residues in wheat DNA was studied in isolated embryos during the first 30 hrs of germination. L-Methionine, but also L-serine serve as methyl group donors in vivo . DNA methylation reaches high values after the maximum of DNA replication at 18 hrs. Isolated nuclei contain S-adenosyl methionine-dependent methyltransferase activity at all stages of germination, a significant increase is observed after the 22nd hour. The plant enzyme can methylate thymus DNA in vitro , it is inhibited by S-adenosyl methionine analogs.

Action of decarboxylated S-adenosylmethionine analogs in the spermidine-synthesizing system from rat prostate

Synthetic decarboxylated S-adenosylmethionine and its nine analogs, as well as four putrescine analogs, were examined in a polyamine-synthesizing system based on rat prostate spermidine synthase. In addition to the natural substrate, decarboxylated S-adenosylmethionine, S-5′-deoxyadenosyl-(5′)-3-ethylthiopropylamine, S-5′-deoxyadenosyl-(5′)-3-propylthiopropylamine, and S-5′-deoxyadenosyl-(5′)-3-butylthiopropylamine were all found to work as the aminopropyl donor to putrescine. Experiments with other analogs of decarboxylated S-adenosylmethionine indicated that the enzyme will transfer only the aminopropyl group, not an aminoethyl or aminobutyl group. The mammalian spermidine synthase could use only one of the two diastereoisomers of decarboxylated S-adenosylmethionine as the aminopropyl donor. The synthase could use 1,4-diamino-2-butene in addition to putrescine as the acceptor of the aminopropyl group from decarboxylated S-adenosylmethionine, but could not use 1,3-diaminopropane or cadaverine. Seven decarboxylated S-adenosylmethionine analogs were inhibitory to spermidine synthesis to varying degrees. Of these analogs, S-5′-deoxyadenosyl-(5′)-2-methylthioethylamine was the most potent inhibitor. Kinetic studies with some of the above compounds were also carried out.

Metabolic formation of nucleoside-modified analogues of S-adenosylmethionine

Evidence is presented for the metabolism of six different adenosine analogues (formycin, 7-deazaadenosine, 8-azaadenosine, 2-fluoroadenosine, purine ribonucleoside and 3′-deoxyadenosine) to their corresponding S-nucleosidylmethionine derivatives in human erythrocytes and mouse lymphocytes. The identification of these novel metabolites was based upon chromatographic and enzymatic characterization of the unique radioactive substance found in cells incubated with each of these adenosine analogues plus L-[methyl- 3H]methionine. The metabolic formation of analogues of S-adenosylmethionine may contribute to the inhibition of cellular methylation reactions caused by various purine analogues.

Affinity chromatography of an S-adenosylmethionine-dependent methyltransferase using immobilized S-adenosylhomocysteine : Purification of the indolethylamine N-methyltransferases of Phalaris Tuberosa

In the cases that have been studied so far, S-adenosylhomocysteine (SAH) is a powerful inhibitor of S-adenosylmethionine (SAM) binding to SAM-dependent methyltransferases. We deduced, from the available data on the binding of SAM and SAH analogues to SAM dependent methyltransferases, that linkage of SAH through the carboxyl group to an immobilized support would lead to a more general affinity adsorbent for SAM-dependent methyltransferases than linkage through other functional groups. This paper describes the synthesis of this affinity adsorbent and its use to purify the two indolethylamine N-methyltransferases of Phalaris tuberosa.

Purification and characterization of rat heart and brain catechol methyltransferase

In an effort to detect the similarities and differences in the properties of rat heart, brain and liver catechol methyltransferase ( S- adenosyl- l-methionine : catechol O-methyltransferase, EC 2.1.1.6), we have determined the cellular distribution of this enzyme activity and extensively purified the soluble and microsomal enzymes present in these tissues. Purification of soluble heart (688-fold) and brain enzymes (240-fold) were achieved using an affinity chromatographic system. The properties of these enzymes were compared with respect to their molecular weights, substrate specificities, inhibitor specificities and immunological properties. The characteristics of the enzyme active sites were investigated using various methyl acceptor substrates and various analogs of S-adenosylmethionine as methyl donors. A series of analogs of S-adenosyl-homocysteine was also evaluated as inhibitors of these enzymes. The immunological properties of the purified soluble and microsomal enzymes from heart and brain were investigated using an antibody isolated from rabbits which had been immunized with the soluble rat liver enzyme. In general the properties of catechol methyltransferases isolated from heart and brain were similar to the properties of the enzyme isolated from liver. Some minor differences in substrate and inhibitor specificities were observed which might suggest slight differences in the active sites of these enzymes.

The S- n-propyl analogue of S-adenosylmethionine

A special strain of Saccharomyces cerevisiae responded to a supplement of S-n- propyl- l- homocysteine in the culture medium by synthesizing S- adenosyl-(S-n- propyl) l- homycysteine , the S- n-propyl analogue of S-adenosylmethionine. S-n- Butyl- l- homocysteine reacted sparingly with this strain, but S- isopropyl- l- homocysteine failed to form detectable quantities of the corresponding S-adenosylsulfonium were compound. The S- n-propyl compound was isolated by extraction of the cells, followed by ion-exchange chromatography, which separated it from endogenous S-adenosylmethionine. The structure was determined by hydrolytic procedures leading to overlapping fragments of known structure, 5′- n-propylthioadenosine and S-n- propyl- l-homocysteine . The new sulfonium compound was examined for its activity as n-propyl donor by substituting it for S-adenosylmethionine in methyltransferase systems. Enzymatic transpropylation was observed with S-adenosylmethionine: l-homocysteine S-methyltransferase (EC 2.1.1.10). Its rate was low in the S-adenosylmethionine: N-acetylserotonin O-methyltransferase system (EC 2.1.1.4), and below recognition with S-adenosylmethionine: guanidonoacetate methyltransferase (EC 21.1.2) and S-adnosylmethionine: histame N-methyltransferase (EC 2.1.1.8).