Isolation and characterization of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a new Lys49 phospholipase A2 homologue.
Myotoxic phospholipases A2 of class II are commonly found in the venoms of crotalid snakes. As an approach to understanding their structure-activity relationship, diverse natural variants have been characterized biochemically and pharmacologically. This study describes a new myotoxic phospholipase A2 homologue, isolated from the venom of Atropoides (Bothrops) nummifer from Costa Rica. A. nummifer myotoxin 1 is a basic protein, with an apparent subunit molecular mass of 16 kDa, which migrates as a dimer in sodium dodecylsulfate-polyacrylamide gel electrophoresis under nonreducing conditions. It is strongly recognized by antibodies generated against Bothrops asper myotoxin II, by enzyme-immunoassay. The toxin induces rapid myonecrosis upon intramuscular injection in mice (evidenced by an early increase in plasma creatine kinase activity), and significant edema in the footpad assay. It also displays cytolytic activity upon cultured murine endothelial cells. The toxin (up to 50 microg) has no detectable phospholipase A2 activity on egg yolk phospholipids, and does not show an anticoagulant effect on sheep platelet-poor plasma in vitro. N-terminal sequence determination (53 amino acid residues) demonstrated that A. nummifer myotoxin II is a new Lys49 variant of the family of myotoxic, class II phospholipases A2. Sequence comparison with other phospholipases A2 revealed Asn53 as a novel substitution. In addition, this myotoxin is the first Lys49 variant presenting Asn in its N-terminus. Consequently, these findings suggest that neither Ser1 or Lys53, usually found in this family of proteins, are essential amino acid residues for their myotoxic, cytolytic, or edema-inducing effects.
Angulo
Y
Y
Instituto Clodomiro Picado, Facultad de Microbiología, Escuela de Medicina, Universidad de Costa Rica, San José.
Olamendi-Portugal
T
T
Possani
L D
LD
Lomonte
B
B
D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003402:Creatine Kinase / Q000097:blood; D003435:Crotalid Venoms / Q000737:chemistry*; D004487:Edema / Q000097:blood / Q000139:chemically induced; D004591:Electrophoresis, Polyacrylamide Gel; D054501:Group II Phospholipases A2; D007273:Injections, Intramuscular; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000187:drug effects / Q000473:pathology; D009336:Necrosis; D010741:Phospholipases A / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D054467:Phospholipases A2; D030162:Reptilian Proteins; D016415:Sequence Alignment
D003435:Crotalid Venoms; D030162:Reptilian Proteins; D007770:L-Lactate Dehydrogenase; D003402:Creatine Kinase; C406786:myotoxin II, Atropoides nummifer; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2