Recombinant rat liver S-adenosyl-L-methionine synthetase tetramers and dimers are in equilibrium.
Rat liver S-adenosyl-L-methionine synthetase is present in two oligomeric forms, tetramers and dimers, with different substrate kinetics and regulation. In vivo the relative amounts of both forms may change in some instances. The basis of this regulatory mechanism is not known. When rat liver cDNA was used to express the protein in Escherichia coli the two oligomeric forms were found. Gel filtration chromatography of the purified recombinant enzyme suggested that these two isoforms might be in equilibrium. This was confirmed by kinetic experiments which showed that the specific activity of the enzyme was dependent on the protein concentration. From these experiments, apparent equilibrium constants of (5.6 +/- 0.4) x 10(5) M-1 and (3.5 +/- 0.9) x 10(5) M-1 were obtained at 2mM and 60 microM methionine concentrations, respectively. Using hydrophobic chromatography on phenyl-Sepharose to separate the tetrameric and dimeric forms, an equilibrium constant of (4.9 +/- 0.7) x 10(5) M-1 was calculated. A rate constant for the dissociation of the tetramer of k-1 = (8.1 +/- 0.4) x 10(-4) s-1 at 4 degrees C was also calculated using the same approach. In summary, we have shown that the rat liver S-adenosyl-L-methionine synthetase produced in bacterial cells is present in two oligomeric forms, tetramers and dimers, which are in equilibrium. This system might be useful for studying the dynamics and the regulation of the distribution of oligomeric forms in the mammalian liver.
Mingorance
J
J
Instituto de Investigaciones Biomédicas, C.S.I.C., Madrid, Spain.
Alvarez
L
L
Pajares
M A
MA
Mato
J M
JM
D000818:Animals; D002850:Chromatography, Gel / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D019281:Dimerization; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008716:Methionine Adenosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D012685:Sepharose
D011994:Recombinant Proteins; D012685:Sepharose; D008716:Methionine Adenosyltransferase