Kinetic Properties and Redox Regulation of Recombinant Calcium-Independent Phospholipase A2γ Reconstituted In Liposomes

Abstract

Calcium-independent phospholipase A2γ (iPLA2γ) catalyses the hydrolysis of fatty acids from phospholipids with the consequent generation of lysophospholipids. iPLA2γ is targeted to peroxisomes and mitochondria and results from our laboratory have shown that the activity of mitochondrial iPLA2γ is increased by a redox-sensitive process [1]. Here we aimed to characterize the properties of isolated recombinant iPLA2γ and test the hypothesis that iPLA2γ is activated by thiol oxidation directly. We expressed His-tagged human iPLA2γ using HepG2 or HEK293 cell lines and reconstituted recombinant purified iPLA2γ into liposomes composed of L-α-Phosphatitylcholine and cardiolipin. Using selected specific fluorescent probes and GC/MS analysis, we observed H2O2-induced release of fatty acids in liposomes containing purified recombinant iPLA2γ but not in liposomes lacking the enzyme. The H2O2-activated enzyme released free fatty acids with a specific activity of about 20 nmol.min-1.mg-1. The activity increased several fold in the presence of bovine serum albumin or in mixed micelles containing non-ionic detergent, indicating a possible inhibition of iPLA2γ by the products of its enzymatic activities. H2O2 activated iPLA2γ with a half-maximal constant corresponding to about 1.5 mol H2O2 per mol of enzyme and the active enzyme was fully inhibited by thiol reducing compounds. These results indicate that the regulation of iPLA2γ is dependent on reversible oxidation of accessible cysteine residues and also on a feedback regulation by either fatty acids or lysophospholipids, which is consistent with the physiological participation of iPLA2γ in cellular redox signaling.Supported by the grant GA15-02051S to M.J. from the Grant Agency of the Czech Republic.1. Jaburek M, Ježek J, Zelenka J, Ježek P. Antioxidant activity by a synergy of redox-sensitive mitochondrial phospholipase A2 and uncoupling protein-2 in lung and spleen. Int. J. Biochem & Cell Biol 45, 816-825, 2013