Purification and properties of phosphatidylinositol-specific phospholipase C from Streptomyces antibioticus

Abstract

A novel type of phosphatidylinositol-specific phospholipase C (PI-PLC) was purified from culture supernatant of a strain of Actinomycetales, Streptomyces antibioticus. The purified enzyme showed a single band on native polyacrylamide gel electrophoresis (native PAGE) with a molecular weight of 32 kDa, but showed two polypeptides, named α- and β-peptides, on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 23 kDa and 15 kDa, respectively. From the results of both eletrophoretic analysis and N-terminal amino acid sequencing, it was estimated that the enzyme was composed of α- and β-peptides. The enzyme could hydrolyze phosphatidylinositol, but not any other glycerophospholipids. The enzyme had pH and temperature optima at around 7.0 and 30°C, respectively, and was stable up to 50°C when incubated at pH 8.0 for 30 min. The PI-PLC was strongly activated by SDS, sodium deoxycholate (SDC) and diethyl ether, but not by Triton X-100, and inhibited by cetylpyridinium chloride (CPC). The enzyme was activated a little by Ca 2+ and was inhibited completely by a chelating agent such as ethylenediaminetetraacetic acid (EDTA) and glycoletherdiaminetetraacetic acid (EGTA). Their inhibitions were restored by the addition of Ca 2+, suggesting that a certain amount of Ca 2+ is essential for the enzymatic activity.