Rat brain protein kinase C: purification, antibody production, and quantification in discrete regions of hippocampus.
Protein kinase C (PKC), a calcium- and phospholipid-dependent kinase, is highly enriched in rat brain, where it may function in signal transduction processes. We purified rat brain PKC to homogeneity by a three-column procedure of diethylaminoethyl-cellulose, phenyl-Sepharose, and protamine-agarose with a yield of 16% and a final specific activity of 9,600 pmol of [3H]phorbol-12,13-dibutyrate bound/mg of protein. The pure protein consisted of a doublet of 80 and 78 kilodaltons. Rabbit antibodies prepared against a beta-type PKC synthetic peptide sequence (RAKIGQGTKAPEEKTANTISK) showed high specificity and sensitivity for PKC and recognized only the 78-kilodalton form of PKC. Micropunches (300 microns in diameter) of rat hippocampal subregions were solubilized in sodium dodecyl sulfate (SDS) sample buffer, electrophoresed on SDS-10% polyacrylamide gels, and transferred to nitrocellulose. PKC was visualized by 125I-protein A autoradiography and quantified by densitometry. The highest concentrations of PKC were found in the CA1 pyramidal cell layer (0.43 +/- 0.04 OD), with the lowest amounts in the CA3 and CA4 pyramidal cell layers (0.11 +/- 0.02 and 0.085 +/- 0.006 OD, respectively). These results demonstrate a simple way of preparing antibodies against domains of PKC. We also describe a procedure for quantifying the relative amounts of PKC in discrete brain regions.
Roth
B L
BL
Department of Psychiatry and Behavioral Sciences, Stanford University Medical Center, California 94305.
Mehegan
J P
JP
Jacobowitz
D M
DM
Robey
F
F
Iadarola
M J
MJ
D000818:Animals; D000917:Antibody Formation; D001921:Brain / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006624:Hippocampus / Q000201:enzymology*; D008297:Male; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000302:isolation & purification*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains
D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C