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.     

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.     

Purification and properties of tRNA(adenine-1)-methyltransferase from rat liver.

An S-adenosylmethionine-dependent tRNA(adenine-1)-methyltransferase has been purified 8,000-fold from rat liver. This preparation gives a single band on polyacrylamide gel electrophoresis and is stable in long term storage. The enzyme has a molecular weight of approximately 95,000. The single methylating capacity of this adenine-1 methyltransferase, using Escherichia coli tRNA2Glu, is methylation of the invariant adenine in the GTpsiC loop. The methylation reaction is dependent on added cation with 20 to 40 mM putrescine being most effective. The Km for S-adenosylmethionine was found to be 0.3 micron, while the Ki for the product inhibitor S-adenosylhomocysteine was 0.85 micron. The Km for tRNAMetf is 12 nM while that for tRNAGlu2 is 33 nM.     

METHYLASES OF MYELIN BASIC PROTEIN AND HISTONE IN RAT BRAIN

—The two enzymes methylating myelin basic protein and histone were purified 170- and 250-fold respectively from the cell sap fraction of rat brain. These enzymes methylated only arginine residues of the two proteins. The enzyme activities were present in all organs tested. Testis has the highest, brain a moderate and liver the lowest activity. Most of the activities were present in the cell sap fraction in brain, liver and testis. Methylation of myelin basic protein and histone was examined in both the cell sap and solubilized nuclear fraction of rat brain during life span after birth. The myelin basic protein methylating activity in the cell sap fraction increased during myelination. Histone methylase from the nuclear fraction was highest at birth and dropped rapidly thereafter. The other activities remained unchanged. The natural occurrence of N^G-mono- and N^G,N^G-dimethylarginine residues in histones prepared from rabbit liver was demonstrated.     

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).     

Modulation by the ratio S-adenosylmethionine/S-adenosylhomocysteine of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. S-Adenosylmethionine/S-adenosylhomocysteine ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio S-adenosylmethionine/S-adenosylhomocysteine can modulate phosphorylation of a specific protein.     

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.     

Rat liver glycine methyltransferase. Cooperative binding of S-adenosylmethionine and loss of cooperativity by removal of a short NH2-terminal segment

Rat liver glycine methyltransferase, a homotetramer, exhibits sigmoidal rate behavior with respect to S-adenosylmethionine (Ogawa, H., and Fujioka, M. (1982) J. Biol. Chem. 257, 3447-3452). The binding experiment shows that the sigmoidicity observed in initial velocity kinetics is explained by the cooperative binding of S-adenosylmethionine to the catalytic sites residing on each subunit. Limited proteolysis of glycine methyltransferase with trypsin in the presence of S-adenosylmethionine yields an enzyme lacking the NH2-terminal 8 residues. The proteolytically modified enzyme retains a tetrameric structure. The truncated enzyme shows no cooperativity with respect to S-adenosylmethionine binding and kinetics. It has values of Vmax and Km for glycine identical to those of the native enzyme, but a 3-fold lower [S]0.5 value for S-adenosylmethionine. The proteolytic modification is without effect on the circular dichroism and fluorescence spectra. Furthermore, the protein fluorescence of the modified enzyme is quenched upon addition of S-adenosylmethionine to the same extent as observed with the native enzyme. These results suggest that a short NH2-terminal segment, which lies outside the active site, is important for communication between subunits.     

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.     

Species differences in the toxicity of p-chloro-o-toluidine to rats and mice. Covalent binding to hepatic macromolecules and hepatic non-parenchymal cell DNA and an investigation of effects upon the incorporation of [3H] thymidine into capillary endothelial cells

The interaction of p-[14C] chloro-o-toluidine with hepatic macromolecules of rats and mice has been investigated. At all time points after single administration the extent of binding decreased in the order protein greater than RNA greater than DNA in both species. The level of binding to mouse liver DNA was greater than that to rat liver DNA after both single and repeated administration. In vitro studies showed that mouse liver fractions catalysed the binding of p-chloro-o-toluidine to calf thymus DNA more readily than rat liver fractions. Conversely, binding to protein and RNA was more marked in the rat than in the mouse. Species differences in DNA repair rates were not observed. The results failed to demonstrate a preferential persistence of binding to mouse liver nonparenchymal cell DNA. Autoradiographic determinations did not demonstrate any effect of p-chloro-o-toluidine upon the incorporation of [3H] thymidine into subcutaneous capillary endothelial cells. The results suggest that different reactive metabolites are responsible for binding to DNA and protein, and that the pattern of reactive metabolites formed from p-chloro-o-toluidine in the mouse differs from that formed in rats.     

Transfer ribonucleic acid methylase activity in adult and foetal rat liver.

Foetal rat liver extracts were found to have higher tRNA methylene activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers. The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100%. The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 muM for Escherichia coli tRNA and 2.1 muM for S-adenosyl-L-methionine. After T1-T2 ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts. It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

Binding of lysophosphatidylcholine to the rat liver fatty acid binding protein

In this study, lysophosphatidylcholine (lysoPC) was shown to bind to a fatty acid binding protein isolated from rat liver. To demonstrate the binding, lysoPC was incorporated into multilamellar liposomes and incubated with protein. For comparison, binding of both lysoPC and fatty acid to liver fatty acid binding protein, albumin, and heart fatty acid binding protein were measured. At conditions where palmitic acid bound to liver fatty acid binding protein and albumin at ligand to protein molar ratios of 2:1 and 5:1, respectively, lysoPC binding occurred at molar ratios of 0.4:1 and 1:1. LysoPC did not bind to heart fatty acid binding protein under conditions where fatty acid bound at a molar ratio of 2:1. Competition experiments between lysoPC and fatty acid to liver fatty acid binding protein indicated separate binding sites for each ligand. An equilibrium dialysis cell was used to demonstrate that liver fatty acid binding protein was capable of transporting lysoPC from liposomes to rat liver microsomes, thereby facilitating its metabolism. These studies suggest that liver fatty acid binding protein may be involved in the intracellular metabolism of lysoPC as well as fatty acids, and that functional differences may exist between rat liver and heart fatty acid binding protein.     

5-methyltetrahydrofolate is bound in intersubunit areas of rat liver folate-binding protein glycine N-methyltransferase

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. It is abundant in the liver, where it uses excess S-adenosylmethionine (AdoMet) to methylate glycine to N-methylglycine (sarcosine) and produces S-adenosylhomocysteine (AdoHcy), thereby controlling the methylating potential of the cell. GNMT also links utilization of preformed methyl groups, in the form of methionine, to their de novo synthesis, because it is inhibited by a specific form of folate, 5-methyltetrahydrofolate. Although the structure of the enzyme has been elucidated by x-ray crystallography of the apoenzyme and in the presence of the substrate, the location of the folate inhibitor in the tetrameric structure has not been identified. We report here for the first time the crystal structure of rat GNMT complexed with 5-methyltetrahydrofolate. In the GNMT-folate complex, two folate binding sites were located in the intersubunit areas of the tetramer. Each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr5, Leu207, and Met215 residues of all subunits. Binding experiments in solution also confirm that one GNMT tetramer binds two folate molecules. For the enzymatic reaction to take place, the N-terminal fragments of GNMT must have a significant degree of conformational freedom to provide access to the active sites. The presence of the folate in this position provides a mechanism for its inhibition.     

Inhibition of glycine N-methyltransferase by 5-methyltetrahydrofolate pentaglutamate

Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the methylation of glycine by S-adenosylmethionine to form sarcosine and S-adenosylhomocysteine. The enzyme was previously shown to be abundant in both the liver and pancreas of the rat, to consist of four identical monomers, and to contain tightly bound folate polyglutamates in vivo. We now report that the inhibition of glycine N-methyltransferase by (6S)-5-CH(3)-H(4)PteGlu(5) is noncompetitive with regard to both S-adenosylmethionine and glycine. The enzyme exhibits strong positive cooperativity with respect to S-adenosylmethionine. Cooperativity increases with increasing concentrations of 5-CH(3)-H(4)PteGlu(5) and is greater at physiological pH than at pH 9.0, the pH optimum. Under the same conditions, cooperativity is much greater for the pancreatic form of the enzyme. The V(max) for the liver form of the enzyme is approximately twice that of the pancreatic enzyme, while K(m) values for each substrate are similar in the liver and pancreatic enzymes. For the liver enzyme, at pH 7.0 half-maximal inhibition is seen at a concentration of about 0.2 microM (6S)-5-CH(3)-H(4)PteGlu(5), while at pH 9.0 this value is increased to about 1 microM. For the liver form of the enzyme, 50% inhibition with respect to S-adenosylmethionine at pH 7.4 occurs at about 0.27 microM. The dissociation constant, K(s), obtained from binding data at pH 7.4 is 0.095. About 1 mol of (6S)-5-CH(3)-H(4)PteGlu(5) was bound per tetramer at pH 7.0, and 1.6 mol were bound at pH 9.0. The degree of binding and inhibition were closely parallel at each pH. At equal concentrations of (6R,6S)- and (6S)-5-CH(3)-H(4)PteGlu(5), the natural (6S) form was about twice as inhibitory. These studies indicate that glycine N-methyltransferase is a highly allosteric enzyme, which is consistent with its role as a regulator of methyl group metabolism in both the liver and the pancreas.     

Identification, purification, and immunochemical characterization of a tocopherol-binding protein in rat liver cytosol.

Tocopherol binding activity accompanying a rat liver cytosolic protein with molecular weight of 30-36 kDa has been demonstrated previously, although the isolation of the protein has not been reported. We now report the purification of an alpha-tocopherol-binding protein (TBP) from rat liver cytosol utilizing three chromatographic procedures: gel filtration, Affi-Gel Blue affinity chromatography, and chromatofocusing. Three peaks of specific alpha-tocopherol-binding activity were resolved on Affi-Gel Blue, referred to as AFB-1A, 1B, and 2. A 32-kDa homogeneous form was obtained after chromatofocusing of AFB-1B. D-alpha-[3H]tocopherol was displaced from homogeneous TBP in the presence of 500-fold excess of nonlabeled alpha-tocopherol, indicating the specificity of the binding. Anti-TBP rabbit antisera identified only one protein in rat hepatic cytosol on Western blotting. TBP immunoreactivity was found in the cytosol of rat liver and the lysate of fractionated hepatocytes, but not in the cytosol of other organs (including the heart, spleen, testes, and lung) nor in the lysate of fractioned Ito cells, endothelial cells, or Kupffer cells isolated from rat liver. Semi-quantitative ELISA demonstrated that rat liver cytosol contained approximately 2 mg TBP/g of cytosol protein. This immunoreactivity was associated with only the 30-36 kDa gel filtration fractions of rat liver cytosol and with both AFB-1A and -1B but not with AFB-2.     

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.     

Selenite: a good inhibitor of rat-liver DNA methylase

DNA methylase from rat liver was partially purified through a DEAE sephacel column and characterized in an in vitro assay with respect to time, protein, DNA and S-adenosylmethionine curves. The Km for S-adenosylmethionine was 2.5 microM. Sodium selenium inhibited the methylation of DNA in a dose dependent fashion when added to the assay. It was also demonstrated that selenite non-competitively inhibits rat-liver DNA methylase with a Ki of 6.7 microM. Dithiothreitol had no effect on selenite inhibition and increasing amounts of DNA did not alter the inhibition. However, increasing amounts of protein overcame the inhibition, suggesting that selenite is reacting with the DNA methylase protein. DNA methylase isolated from selenite treated animals had only 43% of the activity as enzyme from control rats. It appears that selenite is a good inhibitor of DNA methylase.     

Increased content of mRNA for a precursor of S-adenosylmethionine decarboxylase in rat prostate after treatment with 2-difluoromethylornithine

Total poly(A)-containing mRNA was isolated from rat ventral prostate and translated in a reticulocyte lysate system. The proteins corresponding to S-adenosylmethionine decarboxylase were precipitated with a specific antiserum to this protein. Two proteins were found; one having an Mr of 32,000, which corresponded to the subunit of this enzyme, and a larger protein of Mr 37,000. Immunopurification of polysomes with the antiserum to S-adenosylmethionine decarboxylase followed by mRNA extraction yielded an mRNA preparation which was 10-30% pure mRNA for S-adenosylmethionine decarboxylase. The translation of this mRNA showed clearly that the protein of Mr 37,000 was a precursor of the Mr 32,000 S-adenosylmethionine decarboxylase subunit. Treatment with 2-difluoromethylornithine, which depletes cellular spermidine and is known to increase the content of S-adenosylmethionine decarboxylase protein, led to a 9-fold increase in the content of its mRNA, but treatment with methylglyoxal bis(guanylhydrazone) did not change the mRNA content.