Mammalian secreted and cytosolic phospholipase A2 show different specificities for phospholipid molecular species.
Previous studies using phospholipid vesicles containing single molecular species have shown cytosolic phospholipase (85 kDa) (PL) A2 to possess a marked preference for arachidonic acid (20:4n-6)-containing species, while secreted PLA2 (14 kDa) exhibited little acyl chain selectivity. In this study, we have defined the molecular specificity of cytosolic PLA2 using phospholipid vesicles derived from rat liver which 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 and PE vesicles were hydrolysed by either human recombinant cytosolic or porcine pancreatic PLA2. Molecular species compositions were determined by reverse phase high performance liquid chromatography (HPLC) with post-column fluorescence derivitisation. HPLC analysis after limited hydrolysis demonstrated that the secreted phospholipase A2 showed no significant acyl chain specificity using these phospholipid mixtures. However, the cytosolic enzyme demonstrated a high degree of preference for arachidonic acid-containing species such that there was no hydrolysis 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 PE16:0/20:4 and PE18:0/20:4 were hydrolysed to a similar degree. Under these assay conditions, the cytosolic enzyme showed a preference for PE as compared with PC. This study confirms that cytosolic PLA2 is highly selective for sn-2 20:4n-6-containing phospholipid molecular species even when presented with a complex natural species mixture. This specificity is consistent with the cytosolic enzyme having a primary role in the process of arachidonic release within cells.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D005260:Female; D006801:Humans; D008099:Liver / Q000201:enzymology; D010179:Pancreas / Q000201:enzymology; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D005231:Fatty Acids, Unsaturated; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2