Expression and characterization of human group V phospholipase A2.
Group V phospholipase A2 (GV-PLA2) has been shown to be involved in signal transduction and inflammatory processes in cellular studies, but the physical and biochemical properties of this important enzyme have been unclear. We report the over-expression and characterization of GV-PLA2. The GV-PLA2 cDNA was synthesized from human heart polyA+ mRNA by RT-PCR, and an expression construct containing the GV-PLA2 was established. After expression in Escherichia coli cells, the protein was solubilized and purified to homogeneity in a single step using nickel affinity chromatography. The purified GV-PLA2 protein was folded to form active enzyme. The recombinant GV-PLA2 has an absolute requirement for Ca2+ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with sonicated vesicles as substrate. GV-PLA2 preferentially hydrolyzes phosphatidylethanolamine (PE) vesicles compared to phosphatidylcholine (PC) vesicles. However, hydrolysis of PC and PE is equivalent in mixed vesicles of the phospholipids. The fatty acid preference of GV-PLA2 is linoleoyl>palmitoyl>arachidonyl with a PC head group and sonicated vesicles. 3-(3-Actamide-1-benzyl-2-ethylindolyl-5-oxy)propane phosphonic acid (LY311727), a potent inhibitor of human group IIA PLA2, strongly inhibits GV-PLA2 with an IC50 value of about 36 nM which is comparable to its inhibition of group IIA PLA2.
Department of Chemistry and Biochemistry, School of Medicine and Revelle College, University of California, San Diego, La Jolla, CA 92093-0601, USA.
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Chen
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Dennis
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000494:pharmacology; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D017510:Protein Folding; D013379:Substrate Specificity
D007211:Indoles; C095553:LY 311727; D010741:Phospholipases A; D054467:Phospholipases A2