Glutathione synthetase from the fission yeast. Purification and its unique heteromeric subunit structure.
Glutathione (GSH) synthetase (EC 6.3.2.3) was purified from the fission yeast Schizosaccharomyces pombe L972h- and from the GSH synthetase deficient mutant MN101/pYS41, which harbors a plasmid containing the GSH synthetase gene of the fission yeast. GSH synthetase is expressed at 10 times higher the amount in MN101/pYS41 than in wild-type L972h-. The purified enzyme gave a single band on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (native PAGE). The molecular weight of this enzyme was determined to be 1.2 x 10(5) by Sepharose CL-6B gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that this enzyme was composed of two kinds of subunits, A (M(r) = 33 x 10(3)) and B (M(r) = 26 x 10(3)), and existed as a heterotetramer (A2B2). The enzyme purified from the wild-type fission yeast, which did not harbor the plasmid, showed the same electrophoretic mobilities on both native PAGE and SDS-PAGE and similar catalytic properties under standard conditions. This enzyme is most active at 45 degrees C and pH 8.0-8.5 with 20 mM Mg2+ + 10 mM ATP and 50 mM K+. The strict requirement for the monovalent cation is rather specific for the enzymes from yeasts. The presence of sugar components in the enzyme is also observed, similar to that in the rat kidney enzyme.
Nakagawa
C W
CW
Institute for Developmental Research, Aichi, Japan.
Mutoh
N
N
Hayashi
Y
Y
D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology; D001665:Binding Sites; D002241:Carbohydrates / Q000032:analysis; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D018014:Gene Transfer Techniques; D005981:Glutathione Synthase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D007668:Kidney / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009154:Mutation; D010449:Peptide Mapping; D010957:Plasmids; D051381:Rats; D012568:Schizosaccharomyces / Q000201:enzymology*; D012697:Serine Endopeptidases
D002241:Carbohydrates; D046911:Macromolecular Substances; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D005981:Glutathione Synthase