Nonspecific acid phosphatase from Schizosaccharomyces pombe. Purification and physical chemical properties.
Repressible nonspecific acid phosphatase from Schizosaccharomyces pombe was purified to apparent homogeneity, as ascertained from ultracentrifugal, electrophoretic, and chromatographic data. The native protein has a molecular weight of 383,000 as determined by sucrose density gradient centrifugation and 381,000 as determined by gel filtration. The native protein can be dissociated in the presence of 8 M urea-1% sodium dodecyl sulfate into sub-units possessing an approximate molecular weight of 104,000. Neutral sugars account for about 66% of the total molecular weight and contribute to the high solubility and some of the other physical properties of this enzyme. Purified enzyme preparations have a Km for 4-nitrophenyl phosphate of 0.17 mM and a broad substrate specificity, but do not show diesterase activity. Phosphate and sulfate are competitive inhibitors. The enzyme is inactivated at neutral and alkaline pH and at relatively low temperatures. Mannose and galactose was found as the main components of the carbohydrate moiety; glucosamine was present in lower amounts. The amino acid analysis revealed a high content of aspartate, threonine, and serine; no sulfhydryl group could be detected. Pi is released in stoichiometric amount (1 mol per enzyme monomer) on protein digestion.
D000135:Acid Phosphatase / Q000032:analysis* / Q000302:isolation & purification; D000596:Amino Acids / Q000032:analysis; D001203:Ascomycota / Q000201:enzymology*; D001667:Binding, Competitive; D002241:Carbohydrates / Q000032:analysis; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008722:Methods; D008970:Molecular Weight; D010710:Phosphates / Q000032:analysis / Q000494:pharmacology; D012568:Schizosaccharomyces / Q000201:enzymology*; D012995:Solubility; D013329:Structure-Activity Relationship; D013431:Sulfates / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000032:analysis
D000596:Amino Acids; D002241:Carbohydrates; D010710:Phosphates; D013431:Sulfates; D013438:Sulfhydryl Compounds; D000135:Acid Phosphatase