Effect of acylation in the activity of secretory phospholipase A2 acting on human erythrocyte

Abstract

Binding assays with phospholipase A2 from Agkistrodom halys (AhPLA) show a very limited number of high affinity binding sites (5,000). AhPLA does not require calcium for binding, but does so for hydrolysis. In addition, specificity is dependent on enzyme concentration, indicating that such specificity is not due to a specific interaction with a given substrate, but is due to binding at the high affinity site. In the other hand, binding by the non-specific Naja nigricollis PLA (NnPLA) is sensitive to alterations in lipid packing density, such as the addition of lipophilic, cations or anions and osmotic shrinkage, but the AhPLA does not display such sensitivity. In an effort to direct the NnPLA toward specific regions of the human erythrocyte membrane surface, this enzyme was covalently coupled farnesyl pyrophosphonate (FPP) and Methyl arachidonyl fluorophosphonate (MAFP). Approximately, 60 percent of inhibition was observed when this PLA was covalently couple to FPP or MAFP, but a significant increase in specificity toward the 20:4 fatty acids specie was observed with the former. MAFP have an activating effect on the activity of the AhPLA with a small increase in preference for 20:4 species. This phenomenon is due to increase in phospholipid pool size. In addition, FPP and MAFP and was found to have no significant effects on the PLA from Agkistrodon piscivorous. This PLA has an almost identical sequence to that of A. halys, but has no acyl specificity and appears to interact directly with membrane lipid. Work supported by NIH grant 2T34GM07821.