Isolation and characterization of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a new Lys49 phospholipase A(2) homologue

Myotoxic phospholipases A(2) Of class II are commonly found in: the venoms of crotalid snakes. As an approach to understanding their structure-activit y relationship, diverse natural variants: have been characterized biochemic ally and pharmacologically. This study describes a new myotoxic phospholipa se Az homologue, isolated from the venom of Atropoides (Bothrops) nummifer from Costa Rica, A. nummifer myotoxin II is a basic protein, with an appare nt subunit molecular mass of 16 kDa, which migrates as a dimer in sodium do decylsulfate-polyacrylamide gel electrophoresis under nonreducing condition s. It is strongly recognized by antibodies generated against Bothrops asper :myotoxin II, by enzyme-immunoassay. The toxin induces rapid myonecrosis up on intramuscular injection in mice (evidenced by an early increase in plasm a creatine kinase activity), and significant edema in the footpad assay. It also displays cytolytic activity upon cultured murine endothelial cells, T he toxin (up to 50 mu g) has no detectable phospholipase A(2) activity on e gg yolk phospholipids, and does not show an anticoagulant effect on sheep p latelet-poor plasma in vitro. N-terminal sequence determination (53 amino a cid residues) demonstrated that A. nummifer myotoxin II is a new Lys49 vari ant of the family of myotoxic, class II phospholipases A(2). Sequence compa rison with other phospholipases A(2) revealed Asn53 as a novel substitution . In addition, this myotoxin is the first Lys49 variant presenting Asn in i ts N-terminus. Consequently, these findings suggest that neither Ser1 or Ly s53, usually found in this family of proteins, are essential amino acid res idues for their myotoxic, cytolytic, or edema-inducing effects. (C) 1999 El sevier Science Ltd. All rights reserved.