Membrane leakage induced by synergetic action of Lys-49 and Asp-49 Agkistrodon piscivorus piscivorus phospholipases A 2: Implications in their pharmacological activities

Abstract

To understand the mechanism by which Lys-49 phospholipase A 2 from the venom of Agkistrodon piscivorus piscivorus exerts its myotoxic activities, we studied the interactions of this non-catalytic phospholipase A 2 with various phospholipid monolayers and liposomes. We measured the interactions of A. p. piscivorus Lys-49 phospholipase A 2 with phospholipid monolayers in terms of the change in surface pressure of monolayer and with liposomes in terms of the leakage of 6-carboxyfluorescein trapped in liposomes. A. p. piscivorus Lys-49 phospholipase A 2 showed the ability to rapidly and extensively induce the leakage of 6-carboxyfluorescein from anionic liposomes. The extent of leakage was sensitive to the ionic strength of medium and the surface charge density of liposomes, indicating the electrostatic nature of the interactions. Also, this protein was able to penetrate into anionic phospholipid monolayers with surface pressure of up to 38 dyn/cm, indicating the involvement of hydrophobic interactions in its binding to anionic liposomes. Another phospholipase A 2 from the same venom, Asp-49 phospholipase A 2, was shown to assist the lytic activity of Lys-49 phospholipase A 2 toward neutral liposomes by hydrolytically generating anionic patches of reaction products on the liposome surface. These results indicate that Lys-49 phospholipase A 2 binds to and disrupts anionic membranes via both electrostatic and hydrophobic interactions and that two phospholipases A 2 from the same venom work synergetically to enhance their pharmacological activities.