Characterization of phospholipase A2 (PLA2) activity in gerbil brain: enhanced activities of cytosolic, mitochondrial, and microsomal forms after ischemia and reperfusion

Abstract

Brain phospholipase A2 (PLA2) activity has not been well characterized. Given the importance of this enzymatic activity for a variety of cellular functions in the brain, we characterized the subcellular distribution of PLA2 activity in gerbil brain and evaluated how PLA2 activity was altered by ischemia and reperfusion. Cytosolic, mitochondrial, and microsomal fractions were prepared by differential centrifugation of forebrain homogenates. PLA2 activities of each fraction were assayed by measuring release of arachidonic acid (AA) from exogenous 14C-AA-phosphatidylcholine (PC), - phosphatidylethanolamine (PE), and -phosphatidylinositol (PI). Two forms of PLA2 were present in the cytosolic fraction: a high-molecular- weight form, active against PC and PE, and a smaller form with an Mr of approximately 14 kDa, active against PE. In the mitochondrial and microsomal fractions, a single form (Mr approximately 14 kDa) was dominant, active against both PC and PE. The role of PLA2 activation in ischemic brain injury remains controversial. PLA2 enzymatic activity was characterized in gerbil brain after 10 min of common carotid occlusion, followed by 10 min of reperfusion. Ischemic/reperfused brains had significantly higher PLA2 specific activities in each subcellular fraction. Ischemia and reperfusion did not change the gel- filtration elution patterns of PLA2 activity of the various forms of the enzyme. Cytosolic, mitochondrial, and microsomal activities were optimal at a pH of approximately 8.5. Cytosolic PLA2 activity was enhanced when Ca2+ concentration [( Ca2+]) was increased over the physiological range (10(-7) to 10(-6) M). Mitochondrial and microsomal PLA2 activities were also [Ca2+] dependent.