Purification and characterization of neutral trehalase from the yeast ABYS1 mutant.
Neutral trehalase was purified from stationary yeast ABYS1 mutant cells deficient in the vacuolar proteinases A and B and the carboxypeptidases Y and S. The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molecular mass of 160,000 Da on nondenaturing gel electrophoresis and of 80,000 Da on sodium dodecyl sulfate-gel electrophoresis. Maximal activity (114 mumol of trehalose min-1 x mg-1 at 37 degrees C) was observed at pH 6.8-7.0. The apparent Km for trehalose was 34.5 mM. Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose. Neutral trehalase is located in the cytosol. A polyclonal rabbit antiserum raised against neutral trehalase precipitates the enzyme in the presence of protein A. The antiserum does not react with acid trehalase. Dephosphorylation by alkaline phosphatase from Escherichia coli of the active phosphorylated enzyme is accompanied by greater than or equal to 90% inactivation. Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da). The phosphorylated amino acid residue was identified as phosphoserine.
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Biochemisches Institut, Universität Freiburg, Federal Republic of Germany.
Holzer
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D000469:Alkaline Phosphatase / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D004793:Enzyme Reactivators; D004926:Escherichia coli / Q000201:enzymology; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D009154:Mutation; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014198:Trehalase / Q000302:isolation & purification*; D014199:Trehalose / Q000378:metabolism
D004793:Enzyme Reactivators; D004533:Egtazic Acid; D004492:Edetic Acid; D014199:Trehalose; D000469:Alkaline Phosphatase; D014198:Trehalase; D005947:Glucose