Phospholipase A2 activity-independent membrane-damaging effect of notexin

To elucidate whether the phospholipase A2 (PLA2) activity of notexin was exclusively associated with the manifestation of its pharmacological activities, the interaction of notexin with phospholipid liposomes was explored by fluorescence and CD measurement underlying the conditions of depriving its PLA2 activity. Although a higher membrane-damaging activity was noted with Ca2+-bound notexin, abolishment of PLA2 activity by EDTA and Sr2+ could not diminish the membrane-damaging activity of notexin. Fluorescence-quenching studies and CD measurement indicated that Ca2+-bound, Sr2+-bound or metal-free notexin did not adopt the same conformation upon binding with phospholipids. Regardless of the presence of Ca2+, Sr2+ or EDTA, self-quenching assay with rhodamine-labeled notexin revealed that the toxin pertained to form oligomer when it bound with liposomes. Although Lys-modified notexin retained full PLA2 activity, a notable decrease in membrane-damaging activity was observed. These results indicate that notexin could directly cause a leakage of membrane via a PLA2 activity-independent manner, and implicate that another biological event contributes to the activity of notexin in vivo.