Activity of phospholipase C in two-phase systems.
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 CuSO4/H3PO4 or p-methoxybenzaldehyde/H2SO4 reagent. The method is examined for the determination of Vmax and Km values of PCs with varying length acyl chains (C10-C18). The comparison of the kinetic parameters with the Vmax and Km 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.
Haftendorn
Regine
R
Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Halle, Germany.
Ulbrich-Hofmann
Renate
R
D000215:Acylation; D001409:Bacillus cereus / Q000201:enzymology; D002021:Buffers; D002725:Chloroform; D002855:Chromatography, Thin Layer; D003840:Deoxycholic Acid; D004075:Diglycerides / Q000378:metabolism; D004655:Emulsions / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008823:Micelles; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism*
D002021:Buffers; D004075:Diglycerides; D004655:Emulsions; D008823:Micelles; D010713:Phosphatidylcholines; D012996:Solutions; D003840:Deoxycholic Acid; D002725:Chloroform; D010738:Type C Phospholipases