Isolation and characterization of a membrane-associated, calcium-dependent phospholipase A₂ from rabbit lung

Abstract

This paper describes the subcellular localization, solubilization and partial purification of an otherwise tightly membrane-bound phospholipase A₂ in lung. Homogenates of rabbit and dog lung had maximal phospholipase A₂ activity at pH 7.0–7.5 in the presence of 5 mM CaCl₂. Calcium-dependent phospholipase A₂ activity was equally enriched (4–5 fold) in mitochondrial, lysosomal, and microsomal-rich fractions obtained by differential centrifugation of whole lung homogenates. Lacking a specific subcellular localization, the phospholipase A₂ was successfully extracted from whole lung by homogenization in 0.18 N H₂SO₄. The acid extract (pH 1.0) contained 200% of the homogenate’s phospholipase A₂ acitvity and was enriched 3.2 fold. The solubilized phospholipase A₂ was most active at pH 7.0, and was absolutely dependent on and extremely sensitive to low concentrations of CaCl₂. Maximal activity was found with less than 0.5 mM added Ca²⁺; Mg²⁺ and Mn²⁺ would not substitute for Ca²⁺, and EDTA was a potent inhibitor. The phospholipase A₂ showed absolute specificity for the 2-acyl position; lysophospholipase, phospholipase C, and triglyceride lipase activities were not detectable in the acid extract. The enzyme was purified 2924-fold with respect to the homogenate by ion exchange and hydrophobic interaction chromatography. Activity was inhibited to approximately the same extent by indomethacin, mepacrine, and chlorpromazine (ID₅₀=5 × 10⁻⁴ M) but was not inhibited by dexamethasone or aspirin.