Rat Brain Protein Kinase C: Purification, Antibody Production, and Quantification in Discrete Regions of Hippocampus

摘要

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. The pure protein consisted of a doublet of 80 and 78 kilodaltons. Rabbit antibodies prepared against a beta-type PKC synthetic peptide sequence (RAKIGQGTKAPEEK-TANTISK) showed high specificity and sensitivity for PKC and recognized only the 78-kilodalton form of PKC. Micropunches 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 with the lowest amounts in the CA3 and CA4 pyramidal cell layers. 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. Keywords: Phorbolester receptor, Nerve transmission, Neurochemistry, Reprints. (AW)