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Activity of Phospholipase C in Two-Phase Systems

✍ Scribed by Regine Haftendorn; Renate Ulbrich-Hofmann

Publisher
Elsevier Science
Year
2002
Tongue
English
Weight
53 KB
Volume
306
Category
Article
ISSN
0003-2697

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✦ Synopsis

Although phospholipase C (PLC) is known to be activated by water-insoluble organic solvents, most activity assays have been designed to work in an aqueous milieu. Here a sensitive method is described for the determination of PLC activity in two-phase systems. The assay is based on the hydrolysis of phosphatidylcholine (PC) in chloroform/buffer. The initial rates of the reaction are determined by densitometric quantification of the product 1,2-diacylglycerol after its separation by high-performance TLC and staining with a CuSO 4 /H 3 PO 4 or p-methoxybenzaldehyde/H 2 SO 4 reagent. The method is examined for the determination of V max and K m values of PCs with varying length acyl chains (C 10 -C 18 ). The comparison of the kinetic parameters with the V max and K m values of the same substrates in the conventional titrimetric assay, using sodium deoxycholate for micellization of PC, demonstrates the high efficiency of PLC in the two-phase emulsion system.

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