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

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

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca²⁺]_i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of <i>Crotalus atrox</i>. 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 Ca²⁺ influx in human neutrophils without any cytotoxic effect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 μM) and SKF- 96365 (20 μM) significantly inhibited K49-PLA2-induced [Ca²⁺]_i increase. These results suggest that K49-PLA2 modulates [Ca²⁺]_i in human neutrophils via 2-APB- and SKF- 96365-sensitive calcium channels without causing membrane disruption.