GLUTATHIONE SYNTHETASE FROM THE FISSION YEAST - PURIFICATION AND ITS UNIQUE HETEROMERIC SUBUNIT STRUCTURE

Glutathione (GSH) synthetase CEC 6.3.2.3) was purified from the fissio n yeast Schizosaccharomyces pombe L972h(-) and from the GSH synthetase deficient mutant MN10l/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 MN1O1/pYS41 than in wildtype L972h(-) . The purified enzyme gave a single band on polyacrylamide gel electro phoresis in the absence of sodium dodecyl sulfate (native PAGE). The m olecular weight of this enzyme was determined to be 1.2 x 10(5) by Sep harose CL-6B gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that this enzy me 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 (A(2)B(2)). The en zyme purified from the wild-type fission yeast, which did not harbor t he plasmid, showed the same electrophoretic mobilities on both native PAGE and SDS-PAGE and similar catalytic properties under standard cond itions. 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 m onovalent cation is rather specific for the enzymes from yeasts. The p resence of sugar components in the enzyme is also observed, similar to that in the rat kidney enzyme.