Development of an Assay for Phosphatidylinositol-Specific Phospholipase C Activity in T Lymphocytes
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Authors
Taylor, Patricia
Issue Date
1984
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
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.
Description
v, 29 p.
Citation
Publisher
Kalamazoo College
License
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