Interfacial activation, lysophospholipase and transacylase activity of Group VI Ca2+-independent phospholipase A2

Abstract

The Group VI 80-kDa Ca²⁺-independent phospholipase A₂ (iPLA₂) has been purified from murine P388D₁ macrophages and Chinese hamster ovary (CHO) cells. The amino acid sequence of the iPLA₂ has been determined and shown to contain a lipase consensus sequence and eight ankyrin repeats, which makes it distinct from Group I–V PLA₂s. This enzyme appears to play a key role in mediating basal phospholipid remodeling. We now report that the Group VI iPLA₂ displays interfacial activation toward short chain phospholipids, 1-octanoyl-2-heptanoyl-sn-glycero-3-phosphocholine, 1,2-diheptanoyl-sn-glycero-3-phosphocholine, and 1,2-dihexanoyl-sn-glycero-3-phosphocholine micelles. ATP protects the iPLA₂ from a loss in activity as a result of prolonged incubation during the assay. Hence higher enzyme activity is observed in the presence than in the absence of ATP. Similar protection was obtained with glycerol. In addition, the iPLA₂ exhibits multiple activities which are strongly dependent on substrate presentation. The lysophospholipase activity of this enzyme was diminished by Triton X-100 and stimulated by glycerol. With the combination of 50 μM Triton X-100 and 50% glycerol, the enzyme’s lysophospholipase activity achieved equivalent activity to its PLA₂ activity. The iPLA₂ displayed both lysophospholipid/transacylase and phospholipid/transacylase activity, supporting the conclusion that the mechanism of action of iPLA₂ proceeds through an acyl-enzyme intermediate as proposed for the Group IV cPLA₂.