Purification and comparison of two forms of S‐adenosyl‐l‐methionine synthetase from rat liver

Abstract

Only two S-adenosyl-L-methionine synthetase forms exist in rat liver: high-M, S-adenosyl-L-methionine synthetase and low-M, S-adenosyl-L-methionine synthetase, which have been purified to apparent homogeneity as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. High-M, S-adenosyl-L-methionine synthetase had an apparent molecular mass, determined by gel filtration, of 210 kDa and was a tetramer constituted by 48.5-kDa subunits, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The apparent molecular mass of low-M, S-adenosyl-L-methionine synthetase, as estimated by gel filtration, was 110 kDa and was constituted by two subunits of 47 kDa. An antiserum against low-M, S-adenosyl-L-methionine synthetase cross-reacted with the two forms. Reverse-phase HPLC runs of tryptic digestions of high-M, and low-M , S-adenosyl-L-methionine synthetase showed that the peptide maps of the two forms were very similar, if not identical. High-M, S-adenosyl-L-methionine synthetase activity was inhibited by S-adenosyl-L-methionine and pyrophosphate. Depending on the dose used, S-adenosyl-L-methionine activated or inhibited low-M, S-adenosyl-L-methionine synthetase and pyrophosphate had no effect on this form. The two synthetases showed a different specific activity at the physiological concentration of methionine. This report shows that even though the two forms are constructed of the same polypeptide chains, they are regulated in a different manner by methionine and by the products of the reaction.ATP : methionine-S-adenosyl-L-transferase (AdoMet synthetase) catalyzes the formation of AdoMet using methionine and ATP as substrates. Pyrophosphate and phosphate are generated in this reaction [l]. AdoMet is the methyl donor in most transmethylation reactions and also the propylamino donor in the biosynthesis of polyamines [l -31. Of the plasma methionine 48% is metabolized in liver [4] and

Analysis of the 5′ non‐coding region of rat liver S‐adenosylmethionine synthetase mRNA and comparison of the Mr deduced from the cDNA sequence and the purified enzyme

“…Due to the known importance of AdoMet synthetase sulfbydryl groups [14,15] in maintaining the enzyme structure and activity, the high-1W; form of the enzyme was purified as described in [3], to then be chromatographed on a thiopropyl-sepharose column. After its elution with a cysteine gradient, AdoMet synthetase activity was determined (data not shown), the active fractions pooled and a sample loaded on a 10% SDS-PACE gel under reducing conditions.…”

“…In rat liver, two distinct oligomeric forms of the enzyme have been identified and designated as high-and low-Mr AdoMet synthetases. It has been shown that both forms are constituted by the same polypeptide chain, which has an apparent molecular mass of 48.5 kDa on polyacrylamide gel electrophoresis [3,4]. Recently, Horikawa et al [5] have isolated a 1.8 kb cDNAcoding for rat liver AdoMet synthetase.…”

Characterization of Rat Liver-specific Methionine Adenosyltransferase Gene Promoter

“…Hepatic MAT is a cytosolic homo-oligomeric protein, found as a mixture of tetramers and dimers (6,7). Its expression correlates well with liver growth and differentiation, having been proposed to be a marker of the differentiated state of the hepatocyte (8).…”

Differential Effect of Thioacetamide on Hepatic Methionine Adenosyltransferase Expression in the Rat

“…The K m for methionine is lowest for MAT II (Ϸ4-10 µmol/L), intermediate for MAT I (23 µmol/L-1 mmol/L), and highest for MAT III (215 µmol/L-7 mmol/L), with different studies reporting different absolute values. [9][10][11][12][13] The activity of MAT is also modulated by SAM, the product of the reaction it catalyzes. SAM strongly inhibits MAT II (IC 50 ϭ 60 µmol/L), whereas it minimally inhibits MAT I (IC 50 ϭ 400 µmol/L) and stimulates MAT III (up to eightfold at 500 µmol/L SAM).…”

“…8 Different isoforms of MAT differ in kinetic and regulatory properties and responsiveness to sulfhydryl agents and dimethyl sulfoxide (DMSO). 2,[9][10][11][12][13] The kinetic parameters varied in different studies depending on the purification procedure used and the purity of the enzyme. The K m for methionine is lowest for MAT II (Ϸ4-10 µmol/L), intermediate for MAT I (23 µmol/L-1 mmol/L), and highest for MAT III (215 µmol/L-7 mmol/L), with different studies reporting different absolute values.…”