The purification and characterization of a phospholipase A2 activity from the 106 000 × g pellet (microsomal fraction) of bovine brain acting on phosphatidylinositol

Abstract

A phospholipase A2 acting on phosphatidylinositol (PI) has been purified from the 106 000 × g pellet (microsomal fraction) of bovine grey matter. The purification steps included extraction with Triton X-100 (0.05%), ammonium sulfate fractionation (20–50% fraction), consecutive column chromatographic runs on Sephadex G-200 and DEAE-Sephacel, and preparative gel electrophoresis (on 10.5% polyacrylamide gel). These steps achieved a purification of 1614 times. The purified enzyme ran as a single band on sodium dodecyl sulfate (SDS) gel electrophoresis. Molecular weight estimations gave values of 18 300 by SDS gel electrophoresis and 18 521 based on amino acid analysis. Amino acid analysis showed the presence of 173 residues with aspartic acid (46), glutamic acid (26) and glycine (21) being the most abundant. Single residues of cysteine, tyrosine, and arginine were measured. The remaining 11 amino acids were present in amounts ranging from 3 to 11 residues.The purified enzyme had a pH optimum of 7.4, was heat stable (to 70 °C), and was activated by Ca2+ (5 mM). Other divalent cations were either slightly inhibitory (Mg2+ and Mn2+) or strongly inhibitory (Zn2+). The nonionic detergents, Triton X-100 (0.02 to 0.03%) and octyl glucoside (30 mM) showed 70 and 25% stimulations, respectively. Other detergents showed no effect (Cutscum), slight inhibition (G3634A), or strong inhibition (cetyltrimethylammonium bromide). Determination of the apparent Km and Vmax by an Eisthenal–Cornish-Bowden plot gave values of 0.52 mM and 1440 nmol [1-14C]oleic acid min −1∙mg protein −1, respectively, for 1-acyl-2-[1-,14C]oleoyl-sn glycerol-3-phosphoinositol as substrate. The above plot confirmed the presence of a strong inhibition by substrate (i.e., PI) beyond 0.4 mM. The properties of this enzyme and its location (microsomal) make it uniquely different from other phospholipase A2 activities reported for brain. The microsomal location and preference for PI shown by this enzyme lend support to the view that it may function to form lyso-PI in a deacylation–reacylation cycle for altering the fatty acid distribution in PI.