MAMMALIAN SECRETED AND CYTOSOLIC PHOSPHOLIPASE A(2) SHOW DIFFERENT SPECIFICITIES FOR PHOSPHOLIPID MOLECULAR-SPECIES

Previous studies using phospholipid vesicles containing single molecul ar species have shown cytosolic phospholipase (85 kDa) (PL) A(2) to po ssess a marked preference for arachidonic acid (20:4n-6)-containing sp ecies, while secreted PLA(2) (14 kDa) exhibited little acyl chain sele ctivity, Tn this study, we have defined the molecular specificty of cy tosolic PLA(2) using phospholipid vesicles derived from rat liver whic h contain complex mixtures of molecular species, Phosphatidylcholine ( PC) and phosphatidylethanolamine (PE) were isolated from rat liver by chloroform and methanol extraction, and solid-phase separation, PC aci d PE vesicles were hydrolysed by either human recombinant cytosolic or porcine pancreatic PLA(2). Molecular species compositions were determ ined by reverse phase high performance liquid chromatography (HPLC) wi th post-column fluorescence derivitisation. HPLC analysis after limite d hydrolysis demonstrated that the secreted phospholipase A(2) showed no significant acyl chain specificity using these phospholipid mixture s, However, the cytosolic enzyme demonstrated a high degree of prefere nce for arachidonic acid-containing species such that there was no hyd rolysis of other molecular species. The extent of hydrolysis of PC16:0 /20:4 was 1.4-fold greater (P < 0.05, n = 3) than PC18:0/20:4, while P E16:0/20:4 and PE18:0/20:4 were hydrolysed to a similar degree, Under these assay conditions, the cytosolic enzyme showed a preference for P E as compared with PC, This study confirms that cytosolic PLA(2) is hi ghly selective for sn-2 20:4n-6-containing phospholipid molecular spec ies even when presented with a complex natural species mixture, This s pecificity is consistent with the cytosolic enzyme having a primary ro le in the process of arachidonic release within cells. The lack of acy l chain specificity of secreted PLA(2) supports the concept that this enzyme does not have a primary role in this process.