Subcellular characteristics of phospholipase A2 activity in the rat kidney: Enhanced cytosolic, mitochondrial, and microsomal phospholipase A2 enzymatic activity after renal ischemia and reperfusion

Abstract

Phospholipase A2 (PLA2) activities in cytosolic, mitochondrial, and microsomal fractions of rat kidneys were characterized under control conditions, after ischemia, and subsequent to ischemia and reperfusion. Two forms of PLA2 activity were present in the cytosolic fraction: a high molecular weight form, active against phosphatidylcholine (PC), and phosphatidylethanolamine (PE), which upon purification has a molecular mass of 110 kD; and a smaller form (M, ≈ 14 kD), active against PE. In mitochondrial and microsomal fractions a single form (M, ≈ 14 kD), active against both PC and PE, was dominant. Activities in each fraction were optimal at pH 8.5-9.5. Cytosolic PLA2 activity was enhanced when Ca2+ concentration ([Ca2+]) was increased over the range of 10-7 to 10-6 M. Mitochondrial PLA2 activity required higher [Ca2+] for activation (> 10-6 M). After 45 min of ischemia cytosolic PLA2 activity was decreased, whereas mitochondrial and microsomal activities were increased. When ischemia was followed by 1 h of reperfusion, cytosolic, mitochondrial, and microsomal activities were enhanced. Ischemia alone did not change the gel filtration chromatography patterns of PLA2 activity, but ischemia and reperfusion resulted in the appearance of a new peak of activity in cytosolic and mitochondrial fractions (M, ≈ 2-3 kD). Thus, the rat kidney has multiple forms of PLA2 activity, likely representing distinct enzymes, with Ca2+ dependencies suggesting regulation by Ca2+ in vivo. Ischemia and reperfusion result in stable increases of PLA2 activity in each subcellular fraction, perhaps related to covalent modifications of PLA2's, which likely account for membrane phospholipid degradation, and increased tissue levels of unsaturated free fatty acids.