Multiple species of mammalian S-adenosylmethionine synthetase. Partial purification and characterization

Abstract

Two species of S-adenosylmethionine (S-Ado-Met) synthetase (EC 2.5.1.6) exist in rat liver cytosol and a distinct species of the enzyme exists in kidney cytosol. S-Ado-Met synthetases alpha and beta in rat liver cytosol have been partially purified about 200- and 80-fold, respectively. The apparent molecular weight estimated by gel filtration and the sedimentation coefficient are 210 000 and 9 S for S-Ado-Met synthetase alpha and 160 000 and 5.5 S for S-Ado Met synthetase beta. Both enzymes absolutely require Mg2+ and K+ for the activity and are completely inhibited by p-(chloromercuri)-benzoate. Kinetic studies indicate that S-Ado-Met synthetases alpha and beta exhibit negative cooperativity with low S0.5 (ligand concentration required for half-maximal velocity) for L-methionine (17 microM) and ATP (0.5 mM) and positive cooperativity with much higher S0.5 values (S0.5 (L-methionine) = 0.5 mM, S0.5 (ATP) = 2 mM), respectively. The cryoprotectants dimethyl sulfoxide and glycerol markedly lower the S0.5 values of S-Ado-Met synthetase beta without significant effect on Vmax. A single species of S-Ado-Met synthetase has been purified about 1000-fold from rat kidney cytosol. The kidney enzyme, termed S-Ado-Met synthetase gamma, has an apparent molecular weight of 190 000 and a sedimentation coefficient of 7.5 S and is resistant to the inhibition by p-(chloromercuri)benzoate. S-Ado-Met synthetase gamma exhibits slightly negative cooperativity with an apparent S0.5 value for L-methionine of 6 microM and for ATP of 70 microM.

S‐Adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus.

“…The apparent Michaelis-Menten constants obtained by Lineweaver-Burk plots are reported in Table 2. The K , values calculated are in good agreement with those reported in the literature for mammalian CI and y forms [20,22,23] and human lymphocyte AdoMet synthetase [19]. Both double-reciprocal plots showed good linearity indicating the absence of cooperativity, in agreement with the data reported for the enzyme from E. coli [17], human lymphocytes [19] and erythrocytes [16].…”

“…This finding implies that inorganic tripolyphosphate could be an enzyme-bound intermediate which is cleaved by a tripolyphosphatase intrinsically associated with AdoMet synthetase [12-141. AdoMet synthetase is distributed in both prokaryotic and eukaryotic organisms [5,15, 161 and it has been purified to homogeneity from Escherichiu coli [17] [20,23, 241. There is a general agreement that the y form is the major, if not the only form, in normal nonhepatic tissues of rat and man [20,23, 251.…”

“…AdoMet synthetase is distributed in both prokaryotic and eukaryotic organisms [5,15, 161 and it has been purified to homogeneity from Escherichiu coli [17] [20,23, 241. There is a general agreement that the y form is the major, if not the only form, in normal nonhepatic tissues of rat and man [20,23, 251. It is interesting to note, in this respect, that the antiserum against the j l form was shown to cross-react with the a form but not with the y form [22].…”

“…This finding implies that inorganic tripolyphosphate could be an enzyme-bound intermediate which is cleaved by a tripolyphosphatase intrinsically associated with AdoMet synthetase [12-141. AdoMet synthetase is distributed in both prokaryotic and eukaryotic organisms [5,15,161 and it has been purified to homogeneity from Escherichiu coli [17], yeast [18], human lymphocytes [19], rat liver [20 -221 and rat kidney [23]. Several multiple isoforms of the enzyme with different kinetic regulatory and physical properties have been demonstrated in eukaryotes; two forms have been purified in yeast [18] and three isoforms, i.e.…”

“…Several multiple isoforms of the enzyme with different kinetic regulatory and physical properties have been demonstrated in eukaryotes; two forms have been purified in yeast [18] and three isoforms, i.e. a, p, and y [23], have been isolated and characterized in mammalian tissues [20,23,241. There is a general agreement that the y form is the major, if not the only form, in normal nonhepatic tissues of rat and man [20, 23, 251.…”