PROTEIN-KINASE-C IN RAT CEREBRAL MICROVESSELS

Activation of protein kinase C is a key event in the transduction of r eceptor-mediated extracellular signals. Little is known about the role of protein kinase C in the microcirculation of the brain. In this stu dy, we examined protein kinase C in isolated cerebral microvessels. A technique for partial purification of protein kinase C from microvesse ls was employed, using Q-Sepharose batch adsorption and single-step sa lt elution in microfuge tubes. This procedure greatly reduced variabil ity and increased protein kinase C specific activity in both the cytos olic and particulate fractions by nearly 50-fold. The identity of the enzyme was confirmed by its inhibition by staurosporine and bisindolyl maleimide and by its translocation in response to phorbol ester. The l evel of protein kinase C was assessed by [H-3]phorbol ester binding an d the endogenous substrates evaluated by in vitro phosphorylation stud ies. Finally, western blot analysis of protein kinase C isoforms indic ated that the beta-isoform was present in both cytosolic and particula te fractions. The alpha-isoform was present at low levels in the cytos olic fraction, whereas the gamma-isoform was not detected.