Cloning and expression of cystolic phospholipase A2 (cPLA2) and a naturally occurring variant. Phosphorylation of Ser505 of recombinant cPLA2 by p42 mitogen-activated protein kinase results in an increase in specific activity.

Full-length cytosolic phospholipase A2 (cPLA2) was cloned from U937 cells and polymorphonuclear leukocytes (PMNLs) while a naturally occurring variant of cPLA2, which lacks residues Val473-Ala749 but has a C-terminal extension of ILMNLSEYMLWMSKVKRFM (DcPLA2) was cloned from PMNLs and mononuclear leukocytes. We were unable to clone DcPLA2 from U937 cells. When cPLA2 and DcPLA2 were expressed in insect cells, both proteins were detected in cell lysates by SDS/PAGE as single bands of apparent molecular masses 100 kDa and 57 kDa, respectively. Full-length cPLA2 was phosphorylated stoichiometrically by p42 mitogen-activated protein (MAP) kinase in vitro at a similar rate to other physiological substrates of this protein kinase and the major site of phosphorylation was identified by amino acid sequencing as Ser505. [32P]Ser(P)505 in cPLA2 was only dephosphorylated at a slow rate by mammalian tissue homogenates. Protein phosphatases 2A, 2B and 2C all contributed significantly to the overall dephosphorylation of cPLA2. The phosphorylation of cPLA2 by p42 MAP kinase correlated with an approximately 1.5-fold increase in specific enzyme activity which was reversed by dephosphorylation.

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)

The properties of a cloned human high-molecular-mass cytosolic phospholipase A2 investigated using a continuous fluorescence displacement assay: evidence for enzyme clustering on phospholipid vesicles.

The 85 kDa human cytosolic phospholipase A2 has been cloned and expressed in insect Sf21 cells. The pure enzyme has been investigated using a fluorescence displacement assay that provides a continuous record of phospholipid hydrolysis [Wilton (1990) Biochem. J. 266, 435-439]. The unusual kinetic properties of this enzyme, previously described using radioactive assays, were readily demonstrated using the continuous fluorescence assay and were examined in detail. It is proposed that the enzyme clusters on the surface of a fixed number of substrate vesicles during the initial stages of catalysis and that the characteristic burst phase of hydrolysis represents the hydrolysis of these vesicles. This clustering produced a molar ratio of total phospholipid substrate to enzyme of about 450:1 at vesicle saturation with enzyme. Under limiting substrate conditions, the lower secondary rate that is observed results eventually in almost complete hydrolysis of the phospholipid; this was confirmed using radioactive substrate. Evidence is presented that during the initial burst phase, equivalent to hydrolysis of the outer monolayer of the vesicle, the enzyme remains tightly bound but is released as the reaction proceeds towards complete hydrolysis of the phospholipid substrate. In the presence of excess substrate, about 370 mol of fatty acid are released per mol of enzyme during the burst phase and it is calculated that this value also approximates to hydrolysis of the outer monolayer of the vesicle. It is proposed that the formation of a stable enzyme-vesicle complex during the burst phase of phospholipid hydrolysis may be due, at least in part, to protein-protein interactions between adjacent enzyme molecules in order to account for the clustering phenomenon.