Abstract
We describe a simple turbidometric assay for phosphatidylcholine-specific phospholipase C (PC-PLC) (EC 3.1.4.3), phosphatidylinositol-specific phospholipase C (PI-PLC) (EC 3.1.4.10), and sphingomyelinase (SMase) (EC 3.1.4.12), suitable for high-volume screening using unmodified substrates. Under the conditions described, 1 to 10 U/ml of PC-PLC ( Bacillus cereus) induces a rapid and continuous increase in turbidity (0.4 to 0.6 AU at 410 nm) of phosphatidylcholine vesicles (1-10 mM) that highly correlates with hydrolysis. Turbidity increases with the formation of small homogenous particles, which if enzyme and substrate dependent. Analogously, PI-PLC (1-10 U/ml) causes a continuous increase in the turbidity of PI vesicles. SMase also causes a continuous increase in PC vesicle turbidity, but unlike like the glycerol phospholipases, SMase causes a discontinuous increase in vesicles of its proper substrate sphingomyelin (SM). After 8-15% hydrolysis, SM vesicles are converted to large heterogeneous particles permitting detection of SMase activity by visual inspection. Thus, turbidity is a useful property to monitor SMases and C-type phospholipases that cleave vesicle-forming phospholipids. Furthermore, the assay is designed for the microtiter plate format, enabling the continuous and simultaneous monitoring of up to 96 wells.
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