BACTERIAL EXPRESSION AND CHARACTERIZATION OF HUMAN SECRETORY CLASS-V PHOSPHOLIPASE-A(2)

Mammalian secretory class V phospholipase A(2) (PLA(2)) is a newly dis covered PLA(2) that is implicated in eicosanoid formation in inflammat ory cells. As a first step towards understanding the structure, functi on and regulation of this PLA(2), we constructed a bacterial expressio n vector for human secretory class V PLA(2) (hV-PLA(2)), over-expresse d and purified the protein, and determined its physical and kinetic pr operties. When compared with human class IIa enzyme (hIIa-PLA(2)), hV- PLA(2) has several distinct properties. First, hV-PLA(2) can catalyse the hydrolysis of phosphatidylcholine more effectively than hIIa-PLA(2 ) by two orders of magnitude. Secondly, hV-PLA(2) has much higher bind ing affinity and activity for compactly packed phosphatidylcholine bil ayers than hIIa-PLA(2). Finally, hV-PLA(2) has much reduced thermal st ability compared with hIIa-PLA(2). These data suggest that hV-PLA(2) i s better suited than hIIa-PLA(2) for acting on the outer cellular memb rane and liberating arachidonic acid from membrane phospholipids. Also , the unusually low thermal stability of hV-PLA(2) might contribute to tighter regulation of its activities in extracellular media.