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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 18, Number 7
BKCSDE 18(7)
July 20, 1997 

 
Title
Liposome-Based Assay for Phospholipase C
Author
Soo-Jeong Lim, Ui-Chan Koh, Eun-Ok Lee, Chong-Kook Kim*
Keywords
Abstract
Phospholipase C from Clostridium perfringens is known to catalyze the hydrolysis of phospholipids in biological membranes. In this study, a simple and sensitive method for assaying phospholipase C was developed by using liposomes entrapping calcein as a fluorescent marker. Phospholipase C-induced lysis of liposomes was determined by measuring the fluorescence intensity of calcein released out from liposomes, Various liposomes with different compositions were prepared by reverse-phase evaporation method to investigate the effect of liposomal composition on the lytic activity of phospholipase C. The calcein-entrapping efficiency of liposomes was affected by the chain length of fatty acid in phosphatidylcholine constituting liposomes. The lytic activity of phospholipase C was the highest against liposomes prepared with eggPC. The lytic activity decreased with increasing chain length of fatty acid in phosphatidylcholine. Incorporation of cholesterol more than 20% into the liposomal bilayer inhibited the phospholipase C-induced lysis. The lysis of liposomes was more greatly increased by the addition of 10 mM of calcium. The lytic activity of phospholipase C was also affected by the surface charge of liposomes. Taken together, it was concluded that reverse-phase evaporation vesicles composed of dipalmitoylphosphatidylcholine and cholesterol in the molar ratio of 9 : 1 allowed to detect the lowest concentration of phospholipase C (0.10 μ g/assay volume). This study suggested that the use of liposomes can provide a simple, sensitive and inexpensive method for assaying phospholipase C.
Page
761 - 766
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