Development of an Assay for Phosphatidylinositol-Specific Phospholipase C Activity in T Lymphocytes
MetadataShow full item record
Phosphatidylinositol (PI) turnover in phytohemaglutinin (PHA) activated peripheral blood lymphocytes is inhibited by plasma lipoproteins. The most potent are those of the low density classes which have hydrated densities of less than 1.063 g/cm3: very low density lipoproteins d<1.006, intermediate density lipoproteins d=1.006 - 1.019, and low density lipoproteins d=1.019 - 1.063 g/cm3 . Low density (LD) lipoproteins transport most of the cholesterol in the blood and regulate sterol biosynthesis in lymphocytes and other non-hepatic cells. PHA stimulates phosphate incorporation into PI, phosphatidylinositol phosphate (DPI), and phosphatidylinositol bis phosphate (TPI). However, LD lipoproteins block phosphate incorporation into PI only. The susceptible step in PI turnover is the release of phosphoinositol from PI by the action of phospholipase C (PL-C). PL-C splits PI into diacylglycerol and a mixture of inositol-1phosphate and inositol 1-2 cyclic phosphate. An assay was developed to measure PL-C activity using commercial PL-C and was then tested on the soluble fraction of lysed T cells. Enzyme and 14C-inositol labeled PI were reacted together and the soluble portion extracted. Activity was measured as a loss of radioactivity from the organic phase (ie. breakdown of PI into water soluble products). The assay proved to be an effective measure of activity for the commercial PL-C, but a dose-response was not obtained from the soluble fraction of T cells.