Phospholipase A2fromNaja naja sputatrixVenom Is a Muscarinic Acetylcholine Receptor Inhibitor

Abstract

A variety of snake venoms was tested for the ability to inhibit the binding of antagonists to specific muscarinic acetylcholine receptors (mAChRs); the highest activity was found in the venom ofNaja naja sputatrix.The active principle in this venom was isolated by column chromatography on Sephadex G-50, Sephadex G-150, and CM-Sephadex C-25. The final preparation was homogeneous as determined by polyacrylamide gel electrophoresis and HPLC; about sevenfold purification was achieved with a yield of 12%. The isolated active component, which was designated “muscarinic inhibitor,” was found to displace various antagonists from rat synaptosomal membranes, which contain all subtypes of mAChRs. The m1 and m2 recombinant human receptors were also competitive withN. naja sputatrixmuscarinic inhibitor. This antagonist-displacing action was dose dependent, but was independent of the reaction temperature. The isolated muscarinic inhibitor was determined to be a 13.6 kDa, monomeric, neutral protein and to have an N-terminal amino acid sequence which is highly homologous with phospholipase A2from the venoms of genusNaja. N. naja sputatrixmuscarinic inhibitor could hydrolyze phosphatidylcholine in a dose- and temperature-dependent manner. This phospholipase A2enzymatic activity was augmented by the addition of the calcium ion, while it was almost completely abolished by a competitive inhibitor of phospholipase A2enzymes. However, the antagonist-displacing activity was only slightly affected by these agents. The treatment of the muscarinic inhibitor withp-bromophenacyl bromide, which selectively modifies the histidine residue in the catalytic site of a phospholipase A2enzyme, caused complete elimination of both activities. These findings indicate thatN. naja sputatrixmuscarinic inhibitor is a protein with two distinct activities, phospholipase and antagonist displacement, the active centers of which may be in close physical proximity for both actions. This is the first finding that a phospholipase A2is an inhibitor of the muscarinic receptor.