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.

Phospholipase A2 and Its Role in Brain Tissue

“…PLA2 activity was solubilized with 0.05% Triton X-100 and purified using multiple-column chromatographic procedures giving a 1,600-fold purification over crude homogenate. The activity of the purified enzyme was highest with phosphatidylinositol (Ptdlns; 100%), compared with PtdCho (62%), phosphatidic acid (PtdOH; 33%), phosphatidylethanolamine (PtdEtn; 25%), and phosphatidylserine (21%) (Gray and Strickland, 1982). The purified enzyme was stimulated at high Ca 2~concentrations (5 mM) and had a molecular mass of 18.5 kDa and a pH optimum of 7.4 (Table 1) (Gray and Strickland, 1982).…”

“…PLA2 activities were then determined in rat brain synaptosomes and in neuronal and glial cell preparations obtained from rabbit brain (Woelk et al, 1978). Since then, different isoforms of PLA2 have been partially purified and characterized (Gray and Strickland, 1982;Hirashima et al, 1992;Nègre-Aminou and Pfenninger, 1993;Nègre-Aminou et a!., 1996). Ca 2 k-dependent PLA 2 The purification and characterization of PLA2 were started with bovine brain gray matter microsomes.…”

Cytosolic phospholipase A2 is induced in reactive glia following different forms of neurodegeneration

“…The central nervous system (CNS) appears to contain multiple PLA 2 types, including calcium-dependent and calcium-independent forms of various molecular masses and substrate specificities (Gray and Strickland, 1982;Ross et al, 1995;Thomson and Clark, 1995;Hirashima et al, 1992;Yang et al, 1996). Which cells in the brain express which particular PLA 2 isoform is not known.…”

Cloning and Characterization of Novel Mouse and Human Secretory Phospholipase A2s

“…Furthermore, recent studies with mice deficient for both PLA 2 receptor and group IIA sPLA 2 demonstrated a potential role of group IB sPLA 2 /PLA 2 receptor-mediated responses in the progression of endotoxic shock, because the knock-out mice exhibit resistance to endotoxininduced lethality with reduced plasma levels of inflammatory cytokines (37). Besides previously identified sPLA 2 s, other low molecular weight PLA 2 s have been detected in various tissues including the brain and lung (38), suggesting the presence of novel sPLA 2 s that might play a compensatory role for the deficiency in group IIA sPLA 2 or an independently functional role in the inflammatory processes.…”