Purification and characterization of the two constitutively produced acid phosphatase isozymes from Schizophyllum commune

Abstract

The two constitutively produced isozymes (A and B) of acid phosphatase (orthophosphoric monoester phosphohydrolase (acid optimum) EC 3.1.3.2) from Schizophyllum commune were separated and purified. The two isozymes co-purified through acetone precipitation, ion exchange and molecular sieve column chromatography. Ultimately, the isozymes were separated from each other by preparative polyacrylamide gel electrophoresis. This procedure resulted in nearly an 800-fold purification for isozyme A and a 1560-fold purification for isozyme B. The native isozymes had a molecular weight between 340 000 and 370 000, as determined by native polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis revealed that both isozymes apparently were composed of the same two electrophoretically distinguishable subunits having molecular weights of 157 000 and 232 000. The optimum pH for isozyme A was 5·0 and for isozyme B 5·2. Isozyme A had a rather narrow range of substrate specificity, hydrolysing only synthetic phosphomonesters. Isozyme B hydrolysed a broader range of substrates including synthetic phosphomonoesters, nucleotide phosphates and sugar phosphates. The Km values for p-nitrophenyl phosphate for isozymes A and B were 0·11 m m and 0·17 m m , respectively; for α-naphthyl phosphate they were 5·5 m m and 0·95 m m , respectively. The activities of both isozymes were strongly inhibited by low concentrations of fluoride and vanadate. Arsenate and l -(+)-tartrate only weakly inhibited the isozymes' activity at either a low or high concentration. The differences in the properties of these two isozymes may reflect differences in their metabolic roles. Treatment of either isozyme A or isozyme B with a β-1,3-glucanohydrolase caused modifications which resulted in their apparent conversion to the faster-migrating, developmentally regulated isozyme C. Neither the native nor the denatured isozymes showed any difference in migration after treatment with N-glycanase. Because isozyme C can be derived from isozyme A or B, it is hypothesized that production of isozyme C may be under post-translational control.