Acid phosphatase of the yeast Saccharomyces cerevisiae. Purification and properties of the enzyme

Abstract

Acid phosphatase from the yeast Saccharomyces cerevisiae was purified to homogeneity as ascertained by ultracentrifugation and electrophoresis. The purification procedure involved mechanical cell disruption, ethanol precipitation, chromatography on DEAE-cellulose, gel filtration on Sepharose 4B. The sedimentation constant S20^0.580 of the purified enzyme was 15.4 S. Carbohydrate content accounted for 50% of the total molecular weight of the enzyme. The optimum pH for purified enzyme was 3.0-3.5, it was stable at pH 3.0-5.0 at room temperature. After 10 min. incubation at 45° C, 50 per cent of the enzymatic activity was lost. Michaelis constant was found to be 1.3 x 10^-4 M for p-nitrophenylphosphate and 5 x 10^-4 M for 3-glycerophosphate as substrates. The enzyme was inhibited by Hg²⁺, Cu²⁺, Fe³⁺, molybdate, phosphate, arsenate, fluoride ions. Inhibition caused by fluoride ions was noncompetitive, by phosphate - competitive, 5 M urea inactivated the enzyme completely, inactivation was reversible at urea concentration below 2,5 M.