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 515

In this section, we describe the procedure for the measurement of
PKC activity in the cytosolic and membrane fractions of vascular
smooth muscle to enable a comparison of inactive versus activated
PKC. Further, we introduce two methods used to measure PKC
activity; radioimmunoassay ( 14 ) and enzyme-immunoassay ( 11 ).
Radioimmunoassay is a quantitative analysis, used to determine the
absolute activity of PKC. On the other hand, enzyme-immunoassay
is a semiquantitative method, which enables a relative comparison of
PKC activity between samples.

3.2. Sample
Preparation

Cerebral (basilar) arteries are collected using the same procedure
described in Sect. 1.1 . A small piece of the cerebellum is also col-
lected. The tissue is cut into small pieces in ice-cold phosphate
buffered saline (PBS; in mM: 137 NaCl, 2.68 KCl, 8.1 Na 2 HPO 4 ,
1.47 KH 2 PO 4 ), and centrifuged at 250 × g at 4°C for 5 min. Next,
1.0 mL of extraction buffer (containing in mM: 50 Tris, 5 EGTA,
10 benzamide, 0.3% b-mercaptoethanol, 50 mg/mL phehylmeh-
tylsulfonic fl uoride, and 10 mg/mL leupeptine; pH 7.5) is added
to each pellet, and samples are sonicated for 20 s, six times. The
samples are then ultracentrifuged at 100,000 × g for 60 min at 4°C
and the supernatant is collected as the cytosol fraction. The pellet
is then resuspended in the extraction buffer containing 0.1%
Triton-X (1.0 mL), incubated for 60 min on ice, and centrifuged
at 100,000 × g for 60 min at 4°C. This supernatant is used for fol-
lowing experiments as the membrane fraction.

3.3. Measurement
of PKC Activity
(Radioimmunoassay)

With the use of a PKC enzyme assay system (Amersham) and
(g- 32 P)ATP, PKC activity is determined by measuring the incorpo-
ration of 32 P from (g- 32 P)ATP into the substrate-peptide, and
expressed in picomoles of 32 P phosphate transferred per minute.
The samples in each fraction are incubated with the substrate-
peptide in the presence of Ca 2+ , phosphatidylserine, phorbol
myristate, and dithiothreitol under the optimum condition for the
assay system. The reaction is initiated by adding (g- 32 P)ATP to the
mixture at 25°C for 15 min, then terminated by adding the stop
solution provided with the assay kit. The terminated reaction mix-
ture is then spotted on binding paper to separate phosphorylated
substrate-peptide. The binding paper is washed with 5% acetic acid
for 10 min twice, and is placed in the liquid scintillation counter
for 1 min. The amount of PKC in the assay depends on the amount
of tissue used. Therefore, values of PKC activity are normalized by
protein concentration and expressed as “pmol/mg protein/min”.

For enzyme-immunoassay, we use the cytosol fraction of the cere-
bellum as a standard (1, 2, 5, 10, 50, 100, 500, 10 3 , 10 4 , and 10 5
times dilution with the extraction buffer solution; same composition
in radioimmunoassay) to compare PKC activity between samples.
PKC activity is measured using nonradioisotopic protein kinase
assay kit (Medical and Biological Laboratories, Nagoya, Japan).

3.4. Measurement
of PKC Activity
(Enzyme-
Immunoassay)