Bacterial expression and characterization of human secretory class V phospholipase A2.
Mammalian secretory class V phospholipase A2 (PLA2) is a newly discovered PLA2 that is implicated in eicosanoid formation in inflammatory cells. As a first step towards understanding the structure, function and regulation of this PLA2, we constructed a bacterial expression vector for human secretory class V PLA2 (hV-PLA2), over-expressed and purified the protein, and determined its physical and kinetic properties. When compared with human class IIa enzyme (hIIa-PLA2), hV-PLA2 has several distinct properties. First, hV-PLA2 can catalyse the hydrolysis of phosphatidylcholine more effectively than hIIa-PLA2 by two orders of magnitude. Secondly, hV-PLA2 has much higher binding affinity and activity for compactly packed phosphatidylcholine bilayers than hIIa-PLA2. Finally, hV-PLA2 has much reduced thermal stability compared with hIIa-PLA2. These data suggest that hV-PLA2 is better suited than hIIa-PLA2 for acting on the outer cellular membrane and liberating arachidonic acid from membrane phospholipids. Also, the unusually low thermal stability of hV-PLA2 might contribute to tighter regulation of its activities in extracellular media.
Han
S K
SK
Department of Chemistry (M/C 111), University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607-7061, USA.
Yoon
E T
ET
Cho
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D011108:Polymers; D017510:Protein Folding; D011994:Recombinant Proteins; D013379:Substrate Specificity
D008081:Liposomes; D010743:Phospholipids; D011108:Polymers; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2