Biochemical Assessments of Cerebral Vasospasm: Measurement of cGMP, PKC, and PTK in Cerebral Arteries - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 516

Each sample and standard (12 mL each) is mixed with the
reaction mixture (108 mL) provided in the kit using a 96-well
microplate, and incubated at 25°C for 5 min. Then, 100 mL of
mixed sample is transferred to individual wells of 96-well assay
plate coated with a synthetic PKC substrate, and incubated at 25°C
for 5 min. The reaction is stopped by adding stop solution (20%
H 3 PO 4 ), and each well is washed fi ve times with PBS. Biotinylated
mouse monoclonal antibody (100 mL) that detects phosphorylated
serine residues on synthetic PKC substrate is added to each well,
and the plate is incubated at 25°C for 60 min. After washing with
PBS, 100 mL of peroxidase-conjugated streptavidin is added to
each well, and incubated at 25°C for 60 min. After washing again
with PBS, 100 mL of o -phenylenediamine solution, peroxidase sub-
strate, is added and samples are incubated at 25°C for an additional
3-5 min. The reaction is stopped by adding stop solution, and the
absorbance of each well is read at 492 nm by a microplate spectro-
photometer. PKC activity is expressed as a percentage of PKC
activity in the cerebellar cytosol fraction after normalization by
protein concentration.

4. Identifi cation
of PKC Isoforms

PKC is a serine/threonine kinase activated by Ca 2+ and diacylglycerol
(DAG), and 12 distinct isoforms have been cloned and identifi ed
( 13, 17 ). According to their biochemical properties (e.g., struc-
ture, regulator sensitivities), these PKC isoforms are classifi ed into
three groups: conventional, novel, and atypical PKC. Conventional
PKCs (a, b1, b2, and g isoforms) are activated by Ca 2+ and DAG.
Novel PKCs (d, e, h, and q isoforms) are activated by DAG, but
Ca 2+ -insensitive. Atypical PKCs (z, i, l, and m isoforms) are insensi-
tive to both Ca 2+ and DAG, but can be activated by phosphatydil-
serine and ceramides in certain conditions. Additionally, there are
differences between PKC isoforms with regard to substrate speci-
fi city, tissue distribution, and intracellular localization ( 13, 18 ).
Regarding tissue distribution of PKC isoforms, PKCg is dominantly
expressed in the central nervous system, and PKCq is in skeletal
muscle, while PKCa is ubiquitously expressed ( 18 ). This heteroge-
neity of PKC expression is not completely understood, but would
be predicted to contribute to their diverse physiological functions.
In vascular smooth muscle cells, PKC a, b1, b2, d, e, and z iso-
forms are identifi ed ( 15, 19, 20 ). However, these isoforms are
differently expressed in vascular smooth muscle derived from vari-
ous arterial beds (e.g., cerebral, mesenteric). In this section, we
describe a method to identify PKC isoforms expressed in cerebral
arteries using Western blotting.

4.1. Background