ABSTRACT
An improved synthesis of fluorogenic substrate analogues for phosphatidylinositol-specific phospholipase C (PI-PLC) is described. The water-soluble substrates, which are derived from fluorescein, are not fluorescent until cleaved by the enzyme, and provide a convenient means to continuously monitor PI-PLC activity. The improvement in the synthesis lies in the method used to protect the hydroxyl groups of the inositol portion of the substrate molecule and allows a milder deprotection procedure to be used. The result is a much more reproducible synthesis of the substrate. The improved procedure has been employed to synthesize a series of fluorogenic substrates, which differ in the length of the aliphatic tail attached to the fluorescein portion of the molecule. The length of the tail was found to have a significant effect on the rate of cleavage of these substrates.
Subject(s)
Fluorescein/chemistry , Fluorescent Dyes/chemical synthesis , Type C Phospholipases/metabolism , Fluorescein/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Molecular Structure , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase CABSTRACT
The synthesis of a fluorogenic substrate for mammalian phosphoinositide-specific phospholipase C is described. The substrate, based on the widely used fluorescein molecule, is a water-soluble substrate analog of phosphatidylinositol-4-phosphate. The fluorogenic substrate 2 is shown to be a sensitive substrate for human PI-PLC-delta1 in a continuous assay.
