A sensitive assay method for the measurement of S-adenosylmethionine in tissue

An assay method has been developed for the measurement of tissue levels of S-adenosylmethionine based upon the ability of this compound to activate tripolyphosphatase associated with S-adenosylmethionine synthetase beta prepared from rat liver. The method has been used to measure S-adenosylmethionine levels in rat liver after feeding rats on various concentrations of methionine in the diet. The results obtained by this method agree well with those measured by the spectrophotometric method. The limit of sensitivity of the assay was about 0.1 nmol of S-adenosylmethionine in an incubation volume of 0.1 ml (10(-6) M).     

Acetylation of decarboxylated S-adenosylmethionine by mammalian cells

Decarboxylated S-adenosylmethionine was found to be a substrate for the nuclear acetyltransferases that act on polyamines and on histones. The rate of acetylation of decarboxylated S-adenosylmethionine was more than twice that of spermidine at saturating substrate concentrations, and decarboxylated S-adenosylmethionine was an active inhibitor of the acetylation of histones by nuclear extracts from rat liver. The acetylation of decarboxylated S-adenosylmethionine occurred in vivo in SV-3T3 cells exposed to the ornithine decarboxylase inhibitor 2-(difluoromethyl)ornithine. The decline in putrescine and spermidine brought about by exposure to 2-(difluoromethyl)ornithine was found to be accompanied by a large rise in the content of both decarboxylated S-adenosylmethionine and acetylated decarboxylated S-adenosylmethionine. These results indicate that decarboxylated S-adenosylmethionine is metabolized not only in the well-known reactions in which it serves as an aminopropyl donor for polyamine biosynthesis but also by acetylation in reaction with acetyl coenzyme A. Furthermore, the inhibition of histone acetylation by decarboxylated S-adenosylmethionine could contribute to the biological effects brought about by inhibitors of ornithine decarboxylase.     

Detection of proenzyme form of S-adenosylmethionine decarboxylase in extracts from rat prostate

Previous work in which the synthesis of S-adenosylmethionine decarboxylase was studied by translation of its mRNA indicated that it was formed as a proenzyme having a M.W. of about 37,000 that was cleaved to form the enzyme sub-unit of M.W. 32,000 in a putrescine-stimulated reaction. The extent to which the proenzyme accumulates in vivo and is affected by the putrescine concentration was studied by subjecting prostate extracts to Western immunoblotting procedures. The proenzyme form was readily detectable in control prostates (about 4% of the total) and this proportion was increased to 25% when the rats were pretreated for 3 days with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. Conversely, it was decreased to almost undetectable levels after treatment with methylglyoxal bis(guanylhydrazone). These results indicate that the processing of the proenzyme form of S-adenosylmethionine decarboxylase is regulated by the cellular putrescine concentration. This conversion provides another step at which polyamine biosynthesis may be controlled.     

Formation of CO2 from the carboxyl group of S-adenosylmethionine by liver membrane-associated enzymes involves the demethylation-transsulphuration pathway

The activity released from membrane fragments into the supernatant fraction of rat liver homogenate by Triton X-100 and forming ¹⁴CO₂ from carboxyl-labeled S-adenosylmethionine (1) is not a true S-adenosylmethionine decarboxylase. It did not produce decarboxylated S-adenosylmethionine but was also able to use S-adenosylhomocysteine as a substrate. The formation of CO₂ from these two substrates was absolutely dependent on the presence of cytosol proteins and low-molecular weight compounds and it accounted for 5 to 10% of the total S-adenosylmethionine degrading activity of the supernatant fraction. The reaction showed abn initial lag period and was inhibited by every intermediate of the transsulphuration pathway. It is concluded that the formation of CO₂ from S-adenosylmethionine involves the demethylation-transsulphuration route from S-adenosylmethionine to α-ketobutyric acid which is finally decarboxylated.     

Quantitation of S-adenosylmethionine decarboxylase protein

A method for the specific labeling of the active site of S-adenosylmethionine decarboxylase was developed. The method consisted of incubating cell extracts with 3H-decarboxylated S-adenosylmethionine and sodium cyanoborohydride in the presence of a spermidine synthase inhibitor. Under these conditions, S-adenosylmethionine decarboxylase was labeled specifically and stoichiometrically. This procedure was used (a) to establish that the subunit molecular weight of S-adenosylmethionine decarboxylase from rat liver, prostate, and psoas and from mouse SV-3T3 cells was 32 000, (b) to titrate the number of active molecules of S-adenosylmethionine decarboxylase in various cell extracts, and (c) to provide a high specific activity labeled preparation of S-adenosylmethionine decarboxylase for use in radioimmunoassay of this enzyme. Competitive radioimmunoassays using this labeled antigen had a sensitivity such that 3 fmol (0.1 ng) of enzyme protein could be quantitated. The rapid loss of S-adenosylmethionine decarboxylase which occurred when SV-3T3 cells were exposed to exogenous polyamines was shown to be due to a rapid decline in the amount of enzyme protein measured both by titration of the active site and by radioimmunoassay.     

The decarboxylation of S-adenosylmethionine by detergent-treated extracts of rat liver

1. The production of (14)CO(2) from S-adenosyl[carboxyl-(14)C]methionine by rat liver extracts was investigated. It was found that, in addition to the well-known cytosolic putrescine-activated S-adenosylmethionine decarboxylase, an activity carrying out the production of (14)CO(2) could be extracted from a latent, particulate or membrane-bound form by treatment with buffer containing 1% (v/v) Triton X-100 [confirming the report of Sturman (1976) Biochim. Biophys. Acta428, 56-69]. 2. The formation of (14)CO(2) by such detergent-solubilized extracts differed from that by cytosolic S-adenosylmethionine decarboxylase in a number of ways. The reaction by the solubilized extracts did not require putrescine and was not directly proportional to time of incubation or the amount of protein added. Instead, activity a showed a distinct lag period and was much greater when high concentrations of the extracts were used. The cytosolic S-adenosylmethionine decarboxylase was activated by putrescine, showed strict proportionality to protein added and the reaction proceeded at a constant rate. Cytosolic activity was not inhibited by homoserine or by S-adenosylhomocysteine, whereas the Triton-solubilized activity was strongly inhibited. 3. By using an acetone precipitate of Triton-treated homogenates as a source of the activity, it was found that decarboxylated S-adenosylmethionine was not present among the products of the reaction, although 5'-methylthioadenosine and 5-methylthioribose were found. Such extracts were able to produce (14)CO(2) when incubated with [U-(14)C]-homoserine, and (14)CO(2) production was greater when S-adenosyl[carboxyl-(14)C]methionine that had been degraded by heating at pH6 at 100 degrees C for 30min (a procedure known to produce mainly 5'-methylthioadenosine and homoserine lactone) was used as a substrate than when S-adenosyl[carboxyl-(14)C]methionine was used. 4. These results indicate that the Triton-solubilized activity is not a real S-adenosylmethionine decarboxylase, but that (14)CO(2) is produced via a series of reactions involving degradation of the S-adenosyl-[carboxyl-(14)C]methionine. It is probable that this degradation can occur via several pathways. Our results would suggest that part of the reaction occurs via the production of S-adenosylhomocysteine, which can then be converted into 2-oxobutyrate via the transsulphuration pathway, and that part occurs via the production of homoserine by an enzyme converting S-adenosylmethionine into 5'-methylthioadenosine and homoserine lactone.     

Phosphatidylcholine synthesis in castor bean endosperm

Three pathways for phosphatidylcholine synthesis were assayed in castor bean (Ricinus communis var. Hale) endosperm. Phosphatidylethanolamine: S-adenosylmethionine methyl transferase occurred predominantly in the endoplasmic reticulum fraction, but some activity appeared in the mitochondria. Phosphorylcholine glyceride transferase occurred exclusively in the endoplasmic reticulum. The phosphorylcholine glyceride transferase activity was approximately 20-fold greater than the methylation pathway in the endoplasmic reticulum. No exchange activity was found. The Michaelis constant for the methylation was 31 mum for S-adenosylmethionine; phosphatidylethanolamine promoted the reaction slightly while other intermediates stimulated it by about 50%. The pH optimum was 9. Phosphorylcholine glyceride transferase had a Michaelis constant of 9.7 mum for cytidine diphosphate choline but variable results were obtained from diglycerides. The pH optimum was 7.5 and a divalent cation was required, Mg(2+) giving the greatest stimulation.     

S-adenosyl-L-methionine:thioether S-methyltransferase, a new enzyme in sulfur and selenium metabolism

The final urinary excretion product of selenium detoxification is trimethylselenonium ion. An assay has been developed for the enzyme, S-adenosylmethionine:thioether S-methyltransferase, responsible for this final methylation reaction. This assay employed high pressure liquid chromatography separation and quantitation of the trimethylselenonium ion produced by thioether methyltransferase acting on S-adenosylmethionine and dimethyl selenide. The enzyme was shown to reside primarily in the cytosol of mouse lung (30 pmol/mg protein/min) and liver (7 pmol/mg protein/min). Purification from mouse lung to a preparation that exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was achieved by DEAE, gel filtration, and chromatofocusing chromatographies. Thioether methyltransferase is monomeric with a molecular weight of 28,000 and has a pI of 5.3. The pH optimum was 6.3, and Km values for dimethyl selenide and S-adenosylmethionine were 0.4 and 1.0 microM, respectively. The enzyme was inhibited 50% by 25 microM sinefungin, an analog of S-adenosylmethionine, or 40 microM S-adenosylhomocysteine, the reaction product. Pure thioether methyltransferase methylated selenium in dimethyl selenide, tellurium in dimethyl telluride, and S in dimethyl sulfide and many other thioethers. These data suggest a general role for this novel enzyme in the synthesis of onium compounds with increased aqueous solubility helpful in their excretion.     

The role of S-adenosylmethionine in the lysine 2,3-aminomutase reaction

The interconversion of L-lysine and L-3,6-diamino-hexanoate (L-beta-lysine) catalyzed by lysine 2,3-aminomutase is known to be stimulated by added S-adenosylmethionine (Chirpich, T. P., Zappia, V., Costilow, R. N., and Barker, H. A. (1970) J. Biol. Chem. 245, 1778-1789). In this paper we show that enzyme activated by S-[2,8,5'-3H]adenosylmethionine catalyzes the conversion of L-lysine to the equilibrium mixture of L-lysine and L-beta-lysine with incorporation of high levels of tritium into both isomers. The tritium levels in the isomers reflect the equilibrium constant for their interconversion, 84% in the L-beta-lysine and 16% in L-lysine compared with Keq = 5.3 +/- 0.3 in the direction of the formation of L-beta-lysine at pH 7.7 and 30 degrees C. No significant tritium is incorporated into lysine from S-[2,8-3H]adenosylmethionine or S-adenosyl[methyl-3H] methionine under comparable conditions. Therefore, the tritium incorporated into lysine in the former reaction arises from the 5'-position of the 5'-deoxyadenosyl group in S-adenosylmethionine. These experiments implicate the 5'-deoxyadenosyl portion of S-adenosylmethionine in the hydrogen transfer mechanism of this reaction, perhaps in a role analogous to that played by the 5'-deoxyadenosyl moiety of deoxyadenosyl cobalamin in coenzyme B12-dependent rearrangements.     

Effect of methionine deprivation on S-adenosylmethionine decarboxylase of tumour cells

Transference of Walker carcinoma and TLX5 lymphoma from normal L-methionine-containing medium to medium containing limiting amounts of L-methionine, or L-homocysteine only, caused a 2-fold increase of S-adenosylmethionine decarboxylase activity. Kinetic analysis showed an increase in the V value of the enzyme from 22 to 53 pmol/min per mg protein in media containing only 0.1 mM L-homocysteine, without any alteration in the Km value (0.1 mM). The increase in enzyme activity does not result from (a) a reduction of the intracellular level of S-adenosylmethionine, since cycloleucine, an inhibitor of methionine adenosyltransferase, had no effect on enzyme activity; (b) an increase in intracellular adenosine 3',5' monophosphate (cyclic AMP), since high extracellular concentrations of N6-monobutyryl cyclic AMP had no effect on enzyme activity; (c) an alteration of polyamine levels, since addition of micromolar concentrations of exogenous putrescine, spermidine and spermine did not prevent the induction of S-adenosylmethionine decarboxylase activity in methionine-free media containing 0.1 mM L-homocysteine. The increased enzyme activity appears to be mainly due to enhanced stabilization, since the half-life was increased from 2.45 to 5.0 h in media containing only 0.1 mM L-homocysteine. Induction of enzyme activity is specific to the removal of L-methionine, since no increase occurred in the absence of L-serine or L-glycine, or both, or by reduction of the serum concentrations in the medium.     

Purification and characterization of a methyltransferase from Aspergillus parasiticus SRRC 163 involved in aflatoxin biosynthetic pathway

A five step scheme has been developed for the purification of a methyltransferase (MT) from mycelia of 3-day old Aspergillus parasiticus (SRRC 163), which catalyzes one step in the aflatoxin biosynthetic pathway. The S-adenosylmethionine (SAM) requiring MT activity is essential for the conversion of sterigmatocystin (ST) to O-methylsterigmatocystin (OMST) prior to being converted to aflatoxin B1. The purification of the MT was carried out from cell-free extracts by CDR (Cell Debris Remover, a cellulosic weak anion exchanger, Whatman) treatment, QMA ACELL, Hydroxylapatite-Ultrogel, PBE 94 chromatofocusing and FractoGel TSK HW-50F filtration chromatography. The purified enzyme was only about 0.1% of the total extractable proteins. The pI of the protein was about 5.0 as judged by chromatofocusing. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 160-KDa. SDS-polyacrylamide gel electrophoresis revealed two protein subunit bands of molecular masses approximately 110-KDa and 58-KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.87 X 10(4) M-1 cm-1 in 50 mM potassium phosphate buffer (pH 7.5). The reaction catalyzed by the MT was optimum at pH 7.5 and between 25-35 degrees C. The Km of the enzyme for ST and SAM was determined to be 1.8 microM and 42 microM, respectively with an estimated turnover number of the enzyme for ST of 2.2 X 10(-2) per sec.     

Polymethylpolysaccharide synthesis in an ethionine-resistant mutant of Mycobacterium smegmatis.

Mutants of Mycobacterium smegmatis were selected for resistance to ethionine in an effort to obtain methylation-defective strains that were altered in their ability to make methylmannose polysaccharides (MMP) or methylglucose lipopolysaccharides. Two methods were developed for the detection of MMP in cell extracts to aid in the screening for potential mutants, one a qualitative procedure based on iodine binding by the sample after paper chromatography and the other a quantitative procedure based on fluorimetric titration of the MMP with parinaric acid. An ethionine-resistant mutant was obtained that contained only about 25% of the normal level of S-adenosylmethionine and 10% of the normal level of methionine adenosyltransferase (adenosine 5'-triphosphate:L-methionine S-adenosyltransferase, EC 2.5.1.6) activity. When grown in the presence of 0.1% ethionine, the mutant cells contained about 50% of the wild-type levels of methylglucose lipopolysaccharides but only about 7% of the normal level of MMP (wild-type cells contain about 0.14 mM MMP and the mutant contains about 0.01 mM MMP). The amount of fatty acid synthesis in the ethionine-resistant mutant grown in the presence of ethionine was not dramatically altered although the mutant accumulated more short-chain and less long-chain unsaturated fatty acids than the wild-type cells.     

Characterization of an active spore photoproduct lyase, a DNA repair enzyme in the radical S-adenosylmethionine superfamily

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). The enzyme spore photoproduct lyase (SP lyase) has been found to catalyze the repair of SP dimers to thymine monomers in a reaction that requires S-adenosylmethionine. We present here the first detailed characterization of catalytically active SP lyase, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SP lyase is monomeric and is red-brown in color. The purified enzyme contains approximately 3.1 iron and 3.0 acid-labile S(2-) per protein and has a UV-visible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM(-1) cm(-1)) and 450 nm (10.5 mM(-1) cm(-1))). The X-band EPR spectrum of the purified enzyme shows a nearly isotropic signal (g = 2.02) characteristic of a [3Fe-4S]1+ cluster; reduction of SP lyase with dithionite results in the appearance of a new EPR signal (g = 2.03, 1.93, and 1.89) with temperature dependence and g values consistent with its assignment to a [4Fe-4S]1+ cluster. The reduced purified enzyme is active in SP repair, with a specific activity of 0.33 micromol/min/mg. Only a catalytic amount of S-adenosylmethionine is required for DNA repair, and no irreversible cleavage of S-adenosylmethionine into methionine and 5'-deoxyadenosine is observed during the reaction. Label transfer from [5'-3H]S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5'-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP C-6 to generate a substrate radical, and subsequent to radical-mediated beta-scission, a product thymine radical abstracts a hydrogen from 5'-deoxyadenosine to regenerate the 5'-deoxyadenosyl radical. Together, our results support a mechanism in which S-adenosylmethionine acts as a catalytic cofactor, not a substrate, in the DNA repair reaction.     

The kinetic properties and reaction mechanism of histamine methyltransferase from human skin.

The substrate kinetic properties of histamine methyltransferase from human skin were studied at limiting concentrations of both histamine and S-adenosylmethionine. Substrate inhibition by histamine was observed at concentrations above 10 microM. Primary plots showed evidence of a sequential reaction mechanism. The Michaelis constants were derived from secondary plots of slopes from the primary plots ([S]/v versus [S]) versus reciprocal of the second substrate concentration. The mean Km values for histamine and S-adenosylmethionine were 4.2 and 1.8 microM respectively. Histamine in concentrations of 25-100 microM inhibited enzyme activity uncompetitively with respect to S-adenosylmethionine. No substrate inhibition was observed with S-adenosylmethionine. To elucidate the reaction mechanism further, inhibition by the two products, S-adenosylhomocysteine and 1-methylhistamine, was studied. S-Adenosylhomocysteine inhibited non-competitively with respect to histamine and competitively with respect to S-adenosylmethionine. 1-Methylhistamine inhibited non-competitively with respect to histamine and to S-adenosylmethionine. These results are interpreted as providing evidence for an ordered sequential Bi Bi reaction mechanism, with the methyl-group donor S-adenosylmethionine as the first substrate that adds to the enzyme and histamine as the second substrate. 1-Methylhistamine is the first product to leave the enzyme and S-adenosylhomocysteine is the second. The results are discussed in terms of the possible role that this enzyme could play in the modulation of histamine-mediated reactions in skin.     

The transmethylation pathway as a source for adenosine in the isolated guinea-pig heart.

In order to quantify adenosine production from the transmethylation pathway [S-adenosylmethionine (AdoMet)----S-adenosylhomocysteine (AdoHcy) in equilibrium adenosine + L-homocysteine] in the isolated guinea-pig heart under basal conditions (normoxic perfusion with 95% O2) and during elevated adenosine production (hypoxic perfusion with 30% O2), two methods were used. (1) Hearts were perfused with normoxic medium containing [2,5,8-3H]adenosine (5 microM) and L-homocysteine thiolactone (0.1 mM), which brings about net AdoHcy synthesis via reversal of the AdoHcy hydrolase reaction and labels the intracellular pool of AdoHcy. From the decrease in AdoHcy pool size and specific radioactivity of AdoHcy in the post-labelling period, the rate of transmethylation, which is equivalent to the rate of adenosine production, was calculated to be 0.98 nmol/min per g. Adenosine release from the hearts was 40-50 pmol/min per g. (2) Hearts were perfused with hypoxic medium containing [35S]homocysteine (50 microM). Owing to the hypoxia-induced increase in adenosine production, this procedure also results in expansion and labelling of the AdoHcy pool. From the dilution of the specific radioactivity of AdoHcy relative to that of [35S]homocysteine, the rate of AdoHcy synthesis from AdoMet (transmethylation) was calculated to be 1.12 nmol/min per g. It is concluded that in the oxygenated heart the transmethylation pathway is quantitatively an important intracellular source of adenosine, which exceeds the rate of adenosine wash-out by the coronary system by about 15-fold. Most of the adenosine formed by this pathway is re-incorporated into the ATP pool, most likely by adenosine kinase. The transmethylation pathway is essentially O2-independent, and the known hypoxia-induced production of adenosine must be derived from an increase in 5'-AMP hydrolysis.     

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli.     

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.     

Partial purification and characterization of an N2-guanine RNA methyltransferase from chicken embryos.

An N2-guanine RNA methyltransferase has been purified 1000-fold from chick embryo homogenates by phosphocellulose chromatography followed by chromatography on S-adenosylhomocystein-Sepharose. The enzyme was shown to methylate the G10 position of Escherichia coli B tRNAPhe and has a Km of 3X10(-7) M for tRNAPhe and 1.38 X 10(-6) M for S-adenosylmethionine. The molecular weight was estimated to be 77 000 by gel filtration and the pH optimum was 8.0 to 8.5. Magnesium ion was not required for activity but it stimulated the rate of methylation 1.5-fold with an optimum at 12 mM. Ammonium ion stimulated activity about twofold with an optimum at about 83 mM. Sodium and potassium ions above 0.1 M were inhibitory.