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Equilibrium sedimentation experiments of the native acid phosphatase indicate a dimer-tetramer dissociating nonequilibrating system with a dimer Mr = 180,000 g/mol. The hydrolysis of nitrophenylphosphate was used to determine the sedimentation coefficient of the active species. The s20,w value for the species which degrades nitrophenylphosphate is 13.52 +/- 0.46 S in 1% sucrose and 13.72 +/- 0.11 S in 1.3 M sodium chloride, corresponding to the Svedberg value of the tetramer species. Several lines of evidence are presented which, together with previous data, indicate that the Schizosaccharomyces pombe nonspecific acid phosphatase is composed of 4 identical or nearly identical polypeptide chains: a, equilibrium sedimentation analysis of the enzyme in denaturing agents indicates the presence of homogeneous material having Mr = 90,800 g/mol; b, digestion with carboxypeptidase A releases 0.82 mol of tyrosine/monomer molecular weight. Concomitant phosphatase inactivation occurred during the splitting off of the tyrosyl terminal residue. Furthermore, a unique NH2-terminal residue (histidine) was determined.

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.

1. The kinetics of the hydrolysis of nitrophenylphosphate by nonspecific acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2.) from Schizosaccharomices pombe was studied. 2. The kinetic parameters, Km and V, were determined as well as the inhibition constants, K1, for the inhibitors, phosphate and fluoride, as a function of pH. 3. The results, interpreted according to the theories of Dixon and Waley indicated the presence of three ionizable groups on the enzyme itself and one on the enzyme-substrate complex. 4. A model of the hydrolysis of phosphoric acid monoesters by the S. pombe acid phosphatase is proposed based on the ionization state of the reactants and on the results of the inhibition by the competitive inhibitors.