GLYCOSYL-PHOSPHATIDYLINOSITOL ANCHORED ACETYLCHOLINESTERASE AS SUBSTRATE FOR PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C FROM BACILLUS-CEREUS

We investigated the enzymatic properties of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus towards glycosyl-phosphatidylinositol anchored acetylcholinesterase (AChE) from bovine erythrocytes and Torpedo electric organ as substrate. The conversion of membrane form AChE to soluble AChE by PI-PLC depended on the presence of a detergent and of phosphatidylcholine. In presence of mixed micelles containing Triton X-100 (0.05%) and phosphatidylcholine (0.5 mg/ml) the rate of AChE conversion was about 3 times higher than in presence of Triton X-100 alone. Furthermore, inhibition of PI-PLC occurring al Triton X-100 concentrations higher than 0.01% could be prevented by addition of phosphatidylcholine. Ca2+, Mg2+ and sodium chloride had no effect on PI-PLC activity in presence of phosphatidylcholine and Triton X-100, whereas in presence of Triton X-100 alone sodium chloride largely increased the rate of AChE conversion. Determination of kinetic parameters with three different substrates gave K(m)-values of 7-mu-M, 17-mu-M and 2 mM and nu-max-values of 0.095-mu-M.min-1, 0.325-mu-M.min-1 and 56-mu-M.min-1 for Torpedo AChE, bovine erythrocyte AChE and phosphatidylinositol, respectively. The low K(m)-values for both forms of AChE indicated that PI-PLC not only recognized the phosphatidylinositol moiety of the anchor but also other components thereof.