Role of the carbohydrate moiety of phospholipase B from Penicillium notatum in enzyme activity

Two types of phospholipase B from Penicillium notatum—the native enzyme and enzyme modified by endogenous protease (T. Okumura, S. Kimura, and K. Saito (1980) Biochim. Biophys. Acta, 617, 264–273)—were treated with endoglycosidase H (endo-β-N-acetylglucosaminidase H, Streptomyces griseus) to investigate the orientational change of the sugar chains associated with the lower activity of the modified enzyme. On measurement of release of sugar chains, by periodic acid-Schiff staining of endoglycosidase H-treated phospholipase B on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by direct sugar analysis of the isolated endoglycosidase H-treated phospholipase B, distinct curves were obtained for release of sugar chains from the native and modified enzymes with ultimately loss of about 30 and 55%, respectively, of the carbohydrate. Removal of sugar chains from the two enzymes resulted in similar increases in phospholipase B activity (phosphatidylcholine hydrolysis) and their phospholipase A₁ and A₂ activities in the presence of Triton X-100, but no change of lysophospholipase activity (lysophosphatidylcholine hydrolysis). The three former activities of the native and modified enzymes increased to almost 170 and 350%, respectively, of their initial values. However, little increase in phospholipase B activity was observed when the activity was assayed in the absence of Triton X-100, and none when it was assayed in the presence of sodium taurocholate. These findings suggest that the carbohydrate moiety of phospholipase B greatly influence the phospholipase B activity, especially in the presence of Triton X-100, and that the low phospholipase B activity of the modified enzyme is due to excess exposure of sugar chains on the surface of the molecule as a result of protease attack.