Yavin_1995_J.Neurochem_65_2594

The kinetics of protein kinase C (PKC) translocation and down-regulation in the 20-day-old fetal brain following short and long episodes of maternal-fetal blood flow occlusion were examined. Restriction for up to 15 min increased the specific enzymatic activity in the membrane by 73%, indicative of translocation. After a 30-min restriction and a 2.5-h reperfusion the total PKC activity in the cytosol was reduced to approximately 50%, consistent with down-regulation/inactivation. The total membrane PKC activity remained unchanged. Several PKC isoenzymes, including alpha, beta 1, beta 2, epsilon, and zeta, but not gamma, were identified in the fetal brain on western blots using specific antibodies. Compared with postnatal day 15, a greater proportion of the fetal PKC isoforms, particularly alpha and epsilon, were membrane bound, alpha, beta 2, epsilon, and zeta, but not beta 1, were translocated into the membrane compartment after episodes of ischemia alone or ischemia and reperfusion. There were no major identifiable proteolytic fragments in the 50-kDa region. Major losses in the total enzymatic activity were encountered in both cytosol and membrane fractions after storage of the enzyme for 10 days at 4 degrees C. These losses were less profound in membrane fractions from ischemic than control animals, suggesting a relative sparing of activity in the membrane as a result of the insult. Preincubation of DEAE-purified PKC for 30 min at 50 degrees C resulted in enzyme inactivation. This was accompanied by a size reduction (approximately 2-5 kDa) in the gel migration of several isozymes in both cytosol and membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)