Isolation of a cDNA encoding the rat liver S-adenosylmethionine synthetase

We have isolated cDNA clones encoding the rat liver S-adenosylmethionine synthetase by means of immunological screening from a phage lambda gt 11 expression library containing cDNA synthesized from adult rat liver poly(A)-RNA. The amino acid sequence deduced from the cDNA indicates that the rat liver enzyme for this protein contains 397 amino acid residues and has a molecular mass of 43697 Da. The deduced amino acid sequence of rat liver S-adenosylmethionine synthetase was 68% similar to those of yeast S-adenosylmethionine synthetases encoded by two unlinked genes SAM1 and SAM2. The rat liver S-adenosylmethionine synthetase also shows 52% similarity with the deduced amino acid sequence of the MetK gene encoding the S-adenosylmethionine synthetase in Escherichia coli.     

Molecular cloning and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine synthetase.

We previously reported the isolation of a cDNA encoding the liver-specific isozyme of rat S-adenosylmethionine synthetase from a lambda gt11 rat liver cDNA library. Using this cDNA as a probe, we have isolated and sequenced cDNA clones for the rat kidney S-adenosylmethionine synthetase (extrahepatic isoenzyme) from a lambda gt11 rat kidney cDNA library. The complete coding sequence of this enzyme mRNA was obtained from two overlapping cDNA clones. The amino acid sequence deduced from the cDNAs indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,715 Da. The predicted amino acid sequence of this protein shares 85% similarity with that of rat liver S-adenosylmethionine synthetase. This result suggests that kidney and liver isoenzymes may have originated from a common ancestral gene. In addition, comparison of known S-adenosylmethionine synthetase sequences among different species also shows that these proteins have a high degree of similarity. The distribution of kidney- and liver-type S-adenosylmethionine synthetase mRNAs in kidney, liver, brain, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.4-kilobase (kb) mRNA species hybridizable with a probe for kidney S-adenosylmethionine synthetase was found in all tissues examined except for liver, while a 3.4-kb mRNA species hybridizable with a probe for liver S-adenosylmethionine synthetase was only present in the liver. The 3.4-kb kidney-type isozyme mRNA showed the same molecular size as the liver-type isozyme mRNA. Thus, kidney- and liver-type S-adenosylmethionine synthetase isozyme mRNAs were expressed in various tissues with different tissue specificities.     

Regulation of transmethylation by an S-adenosylmethionine binding protein

A protein which has a high affinity for S-adenosylmethionine (SAM) has been partially purified from rat liver. This binding protein stimulates both the rate and extent of product formation when added to both a lipid methylating system, phosphatidylethanolamine: SAM-N-methyltransferase, and an RNA methylating system, the t-RNA methylase complex from rat liver. The S-adenosylmethionine binding protein by itself has no enzymatic activity in either transmethylation system.     

S-adenosylmethionine inhibits ubiquitin-proteasome system in vitro and on rat vascular smooth muscle cells

S-adenosylmethionine is a metabolite regulating many biological processes; S-adenosylmethionine effect on ubiquitin-proteasome system (UPS) has not been studied yet. We investigated S-adenosylmethionine effects on UPS activity both in vitro, by inhibitor screening assay, and in rat vascular smooth muscle cells, by Western Blot of proteasomal targets. We found that S-adenosylmethionine inhibited UPS activity.     

Metabolism of S-adenosylmethionine in rat hepatocytes: transfer of methyl group from S-adenosylmethionine by methyltransferase reactions

Treatment of rats with a methionine diet leads not only to a marked increase of S-adenosylmethionine synthetase in liver, but also to the increase of glycine, guanidoacetate and betaine-homocysteine methyltransferases. The activity of tRNA methyltransferase decreased with the increased amounts of methionine in the diets. However, the activities of phospholipids and S-adenosylmethionine-homocysteine methyltransferases did not show any significant change. When hepatocarcinogenesis induced by 2-fluorenylacetamide progresses, the activities of glycine and guanidoacetate methyltransferases in rat liver decreased, and could not be detected in tumorous area 8 months after treatment. The levels of S-adenosylmethionine in the liver also decreased to levels of one-fifth of control animals at 8 months. The uptake and metabolism of [methyl-3H]-methionine and -S-adenosylmethionine have been investigated by in vivo and isolated hepatocytes. The uptake of methionine and transfer of methyl group to phospholipid in the cells by methionine were remarkably higher than those by S-adenosylmethionine. These results indicate that phospholipids in hepatocytes accept methyl group from S-adenosylmethionine immediately, when it is synthesized from methionine, before mixing its pool in the cells.     

Molecular cloning and nucleotide sequence of cDNA encoding the human liver S-adenosylmethionine synthetase.

cDNA clone for human liver S-adenosylmethionine synthetase (liver-specific isoenzyme) was isolated from a cDNA library of human liver poly(A)+ RNA. The cDNA sequence encoded a polypeptide consisting of 395 amino acid residues with a calculated molecular mass of 43675 Da. Alignment of the predicted amino acid sequence of this protein with that of rat liver S-adenosylmethionine synthetase showed a high degree of similarity. The coding region of the human liver S-adenosylmethionine synthetase cDNA sequence was 89% identical at the nucleotide level and 95% identical at the amino acid level to the sequence for rat liver S-adenosylmethionine synthetase.     

Separation of different forms of S-adenosylmethionine synthetase by fast protein liquid chromatography

A method is described by which different forms of S-adenosylmethionine synthetase are separated. The method makes use of fast protein liquid chromatography on an anion exchange hydrophilic polyether resin. Different forms of S-adenosylmethionine synthetase from rat and human liver and kidney and rat zajdela hepatoma cells can be separated within 15 min. From a mixture of rat liver and kidney cytosols all three forms alpha, beta and gamma can be separated. The time needed to separate the different forms of S-adenosylmethionine synthetase is reduced from 3 h with conventional gel filtration methods, to 15 min using this HPLC anion-exchange method. Also the amount of tissue needed to detect the different forms is reduced from 125 mg to 12.5 mg of fresh rat liver tissue. These advantages make this newly developed method applicable when large numbers of samples have to be analyzed or when only small amounts of tissue are available.     

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.     

Synthesis of S-adenosylmethionine synthetase isozymes from rat and mouse livers. Immunochemical studies

The alpha- and beta-forms of S-adenosylmethionine synthetase in rat liver were completely fractionated by chromatography on a hydrophobic resin, phenyl-Sepharose. The alpha-form was eluted in low-ionic strength buffer, and the beta-form was eluted with 50% dimethylsulfoxide. The alpha-form is less sensitive to dimethylsulfoxide, whereas the beta-form is strikingly stimulated by dimethylsulfoxide, after removal of the dimethylsulfoxide. The levels of the alpha-form activity in rat liver after treatment with ethionine and adenine for 2 consecutive days, and those of the beta-form activity in mouse liver on the 12th day after transplantation of Ehrlich ascites tumor cells, were increased several fold compared to normal liver. Immunochemical titrations with specific antibody against the beta-form as well as kinetic studies indicated that the observed increase in the levels of each activity from the S-adenosylmethionine synthetase isozymes is due to an increase in the cellular content of the enzyme.     

Decreased activities of S-adenosylmethionine synthetase isozymes in hereditary hepatitis in Long-Evans rats

Isozyme patterns of S-adenosylmethionine synthetase have been measured with or without dimethylsulfoxide in liver of LEC rat hereditary hepatitis. The activities of the alpha- and beta-forms are decreased with age after birth, and decreased to a half level of 36 weeks after birth. Concentration of S-adenosylmethionine in the liver is almost a half level of control rat. However, the activity of glycine- and tRNA-methyltransferases in the liver shows no significant change.     

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 purification and kinetic properties of liver microsomal-catechol-o-methyltransferase

A membrane-bound form of catechol-O-methyltransferase (COMT) has been solubilized and partially purified from rat liver microsomes. The purified microsomal-COMT was found to be neutralized by antibody to the soluble-COMT. The pH optimum, the kinetic constants for catechol substrates and S-adenosylmethionine, and the sensitivity to inhibitors of this microsomal-COMT were all found to be similar to the soluble-COMT.     

Purification and properties of glycine N-methyltransferase from rat liver

Glycine N-methyltransferase (EC 2.1.1.20) has been purified to homogeneity from rat liver. The enzyme has a molecular weight of 132,000 by sedimentation equilibrium method. This value is in good agreement with a value of 130,000 obtained by Sephadex G-150 chromatography. The molecular weight of the denatured enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 31,500. The numbers of peptides obtained by tryptic digestion and by cyanogen bromide cleavage are one-fourth of those expected from the contents of lysine plus arginine residues and methionine residues, respectively. By Edman degradation, phenylthiohydantoin-leucine is the only amino acid derivative released from the enzyme. Neither sugar nor phospholipid is detected in the purified preparation. These data indicate that the rat liver glycine N-methyltransferase is a simple protein consisting of 4 identical subunits. The enzyme has an isoelectric pH of 6.4, and is most active at pH 9.0. From the circular dichroism spectrum, an alpha helix content of about 11% is calculated. Whereas the initial velocity as a function of glycine concentration gives a Michaelis-Menten kinetics, the enzyme shows a positive cooperativity with respect to S-adenosylmethionine. The concentrations of glycine and S-adenosylmethionine which give a half-maximum velocity are 0.13 mM and 30 microM, respectively, at pH 7.4 and 25 degrees C.     

Analysis of isethionic acid in mammalian tissues

A gas-liquid chromatographic assay has been developed to measure isethionic acid after methylation with diazomethane. The identity of the products of methylation has been confirmed by mass-spectrometry and nuclear magnetic resonance spectroscopy. The method was used to measure isethionic acid in rat heart, dog heart and rat brain. The assay was validated by measuring isethionic acid on squid axoplasm. We have been able to detect only trace amounts of isethionic acid in rat brain (0.2 mg/100g) and rat heart (0.1 mg/ 100g). None was found in dog heart.     

Partial purification of a ribosomal ribonucleic acid methylase from rat liver nuclei and methylation of undermethylated nuclear ribonucleic acid from regenerating liver of ethionine-treated rat

S-Adenosylmethionine-dependent ribosomal RNA (rRNA) methylase has been purified approx. 90-fold from rat liver nuclei. The partially purified methylase catalyzes the methylation of base and ribose in hypomethylated nuclear rRNA prepared from the regenerating rat liver after treatment with ethionine and adenine. The enzyme has an apparent molecular weight of about 3 x 10(4) and a sedimentation coefficient of 3.0 S. The enzyme is optimally active at pH 9.5 and sensitive to p-chloromercuribenzoate. Thiol-protecting reagents, such as dithiothreitol, are necessary for its activity, and the enzyme requires no divalent cations for its full activity. This enzyme did not efficiently transfer the methyl group to nuclear rRNA from normal rat liver, compared with hypomethylated nuclear rRNA. Methyl groups were mainly incorporated into pre-rRNA larger than 28 S, and the extent of 2'-O-methylation of ribose by this enzyme was greater than that of base methylation in the hypomethylated rRNA. No other nucleic acids, including transfer RNA (tRNA) and microsomal RNA from normal as well as ethionine-treated rat livers, tRNA from Escherichia coli, yeast RNA, and DNA from rat liver and calf thymus, were significantly methylated by this methylase. These results suggest that partially purified rRNA methylase from rat liver nuclei incorporates methyl groups into hypomethylated pre-rRNA from S-adenosylmethionine.     

A radiometric assay for kynurenine 3-hydroxylase based on the release of 3H2O during hydroxylation of L-[3,5-3H]kynurenine.

A rapid and sensitive assay for kynurenine 3-hydroxylase (KH) has been developed. This radiometric assay is based on the enzymatic synthesis of tritiated water from L-[3,5-3H]kynurenine during the hydroxylation reaction. Radiolabeled water is quantified following selective adsorption of the isotopic substrate and its metabolite with activated charcoal. The assay is suitable for detecting 0.1 pmol enzyme activity per minute per milligram protein in tissues displaying low levels of the enzyme. The amount of water produced in the reaction, as calculated from the tritium released, was stoichiometric with the 3-hydroxykynurenine product detected by HPLC. Rat liver KH was characterized by cofactor specificity and kinetic parameters. NADPH was preferred over NADH as coreductant in the reaction. Tetrahydrobiopterin was not a cofactor. The tissue distribution of KH activity in the rat suggested that the majority of active enzyme is located in liver and kidney. Detectable amounts were found in several other tissues, including brain which had low but significant levels of activity in every region assayed.     

Refolding and characterization of rat liver methionine adenosyltransferase from Escherichia coli inclusion bodies

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major methyl donor for transmethylation reactions. Attempts to perform structural studies using rat liver MAT have met with problems because the protein purified from cellular extracts is heterogeneous. Overexpression of the enzyme in Escherichia coli rendered most of the protein as inclusion bodies. These aggregates were purified by specific washes using urea and Triton X-100 and used for refolding. Maximal activity was obtained when chaotropic solubilization included the structural cation Mg(2+), the protein concentration was kept below 0.1 mg/ml, and denaturant removal was carried out in a two-step process, namely, a fast dilution followed by dialysis in the presence of 10 mM DTT or GSH/GSSG redox buffers. Refolding by this procedure generated the oligomeric forms, MAT I and III, which were basically indistinguishable from the purified rat liver forms in secondary structure and catalytic properties.     

Heterogeneity of antibodies to metallothionein isomers and development of a simple enzyme-linked immunosorbent assay

A competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of metallothionein (MT) in tissues and body fluids has been developed. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT-2 polymer, a biotinylated secondary antibody, and peroxidase conjugated avidin. With a 1:4000 dilution of the immunoglobulins, typical standard curves (logit-log regression) provide a linear range of 0.1–100 ng for MT-2 and 10–1000 ng for MT-1. Fifty percent inhibition is accomplished with 15 ng and 250 ng for MT-2 and MT-1, respectively. Rat liver MT-1 and MT-2 containing different metals (Ag, Cu, and Zn) inhibited the antibodies as effectively as CdMT. However, the antibodies exhibited greater affinity for both Apo-MT isoforms. Previously reported discrepancies between results obtained by metal binding assays (e.g., Ag-hem binding) and radioimmunoassay for MT levels in tissues have been largely resolved. By addition of 1% Tween 20 to samples, the ELISA routinely estimated the total MT in samples of rat, mouse, and human liver and kidney at 88% of the value obtained by the silver-hem binding assay. Specific antibodies to MT-2 were purified from our anti-serum by affinity purification using CH-Sepharose 4B coupled with rat liver MT-1. Estimation of MT in samples using purified MT-2 antibodies provided slightly lower values (72%) for MT in tissues as compared to the Ag-hem method. The predominant form of MT in tissues of control animals was found to be MT-2. Therefore, the MT-2 specific antibodies may be useful for the study of the functions of MT isoforms. Levels of total MT in tissues and biological fluids of rats injected with CdCl₂ (0.3 mg Cd/kg) and Cd-MT (0.3 mg Cd/kg) were estimated by ELISA. The results suggest urinary MT levels may be related to kidney damage.     

Analysis of S-adenosylmethionine and related sulfur metabolites in animal tissues

A high-performance liquid chromatographic method was devised that separates S-adenosylmethionine and related sulfur metabolites on a Radial-PAK SCX cation-exchange column using a four-step NH4COOH/(NH4)2SO4 elution gradient. This new procedure permits, in a single run of 60 min, the quantitative analysis of S-adenosylmethionine, S-adenosylhomocysteine (AdoHcy), 5'-deoxy-5'-methylthioadenosine, decarboxylated S-adenosylmethionine, decarboxylated AdoHcy, inosylhomocysteine, and other related metabolites. Furthermore, this method allows the detection in rat tissues of novel sulfur metabolites, S-inosylhomocysteine and decarboxylated AdoHcy. Perturbation of the levels of some of these metabolites could be detected in rat livers and spleens after the administration of 3-deazaadenosine, an inhibitor of AdoHcy hydrolase, but could not be detected in rat adrenal glands. It is notable that decarboxylated AdoHcy disappeared in the livers of rats treated with 3-deazaadenosine. HeLa cells incubated with [35S]methionine displayed the incorporation of the labeled sulfur into S-adenosylmethionine, AdoHcy, decarboxylated S-adenosylmethionine, S-inosylhomocysteine, and 5'-deoxy-5'-methylthioadenosine.     

Effect of aldehydes on polyamine metabolism. III. Inhibition of S-adenosyl methionine decarboxylase (SAMD) by CCl4 and by aldehydes produced during lipid peroxidation

In isolated rat liver cells in which lipid peroxidation is stimulated by CCl4, a strong inhibition of S-adenosylmethionine decarboxylase (SAMD) activity occurs. Some purified aldehydes, which are produced during lipid peroxidation, are able to inhibit SAMD activity in Yoshida hepatoma cells. The most active aldehyde is hydroxypentenal (HPE). It inhibits by 50% SAMD activity at 0.5 mM concentration in entire hepatoma cells, or in hepatoma cell sap, and at 0.1 mM concentration in partially purified hepatoma cell sap fractions.