Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein

We fortuitously observed a human neutrophil intracellular free-cal- cium concentration ((Ca 2+ )i) increasing activity in the commercially available phos- phodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49- phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca 2+ infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 M) and SKF- 96365 (20 μM) signifi cantly inhibited K49-PLA2-induced (Ca 2+ )i increase. These results suggest that K49-PLA2 modulates (Ca 2+ )i in human neutrophils via 2-APB- and SKF- 96365-sensitive calcium channels without causing membrane disruption.