Partial purification and characterization of the interconvertible forms of trehalase from Saccharomyces cerevisiae

Cryptic trehalase from Saccharomyces cerevisiae was purified about 3000-fold. The recovery of 970% of the original “activity” indicated the removal of an inhibitor of the enzyme. Active trehalase, obtained through phosphorylation of cryptic trehalase by cAMP-dependent protein kinase, was isolated by chromatography on DEAE-cellulose. A major phosphorylated protein, with an apparent M r of 86,000, was detected after SDS-polyacrylamide gel electrophoresis. This protein band correlated exactly with the elution profile of trehalase activity and 32 P i incorporation into the enzyme on DEAE-cellulose chromatography. Partially purified active trehalase showed absolute specificity towards trehalose with an apparent K m of 4.79 × 10 −3 m. Both forms of the enzyme showed an apparent molecular weight of 160,000, by gel filtration. Centrifugation on a glycerol density gradient indicated multiple forms of trehalase- c, with M r of 320,000, 160,000, and 80,000. After activation of each of these forms by protein kinase, a single form of trehalase- a was observed, with a M r of 160,000. Trehalase- c appears to be a totally inactive form of the enzyme. The only mechanism of activation seems to be phosphorylation by cAMP-dependent protein kinase. When the protein kinase concentration was varied, at a fixed trehalase- c concentration, a sigmoidal activation plot was obtained. This result suggests the occurrence of multiple forms of cryptic trehalase.