EXPERIMENTAL ANDTOXICOLOGIC PATHOLOGY Experimental and Toxicologic Pathology 57 (2006) 239–245 Paralyzing and myotoxic effects of a recombinant bothropstoxin-I (BthTX-I) on mouse neuromuscular preparations Ma´rcia Gallacci a,Ã , Maristela Oliveira a , Maeli Dal Pai-Silva b , Walter Luı´s Garrido Cavalcante a , Patrick Jack Spencer c a Departamento de Farmacologia, Instituto de Biocieˆncias, Universidade Estadual Paulista, Rubia˜o Ju´nior, Botucatu, CEP 18618-000, Sa˜o Paulo, Brazil b Departamento de Morfologia, Instituto de Biocieˆncias, Universidade Estadual Paulista, Botucatu, Sa˜o Paulo, Brazil c Laborato´rio de Radiobiologia Instituto Pesquisas Energe´ticas e Nucleares, SP, Brazil Received 12 December 2004; accepted 1 September 2005 Abstract As a first step to investigate the structure–function relationship of bothropstoxin-I (BthTX-I), a myotoxin from Bothrops jararacussu snake venom, our group previously cloned a recombinant toxin (rBthTX-I) in Escherichia coli. The aim of this work was to characterize the biological activities of this rBthTX-I (1.0 mM) in both phrenic-diaphragm and extensor digitorum longus preparations in vitro, by means of myographic and morphologic techniques. Native BthTX-I (1.0 mM) was used as a standard. The influence of heparin (27.5 mg/ml) upon the biological activities of both toxins was also investigated. rBthTX-I had similar effects to the native toxin inducing blockage of both directly and indirectly evoked contractions in phrenic-diaphragm preparations, and muscle damage characterized by edema, round fibers, and cell areas devoid of myofibrils. Interestingly the paralyzing activity of rBthTX-I was slightly more potent than the native toxin. Heparin prevented paralyzing and myotoxic effects of both the native and recombinant toxins. This work shows that rBthTX-I was expressed in a fully active form, and presents a biological profile similar to the native toxin. r 2005 Elsevier GmbH. All rights reserved. Keywords: Myotoxin; Phospholipase A 2 ; Muscle damage; Ultrastructure Introduction Myotoxic phospholipases A 2 (PLA 2 s) have been identified in many snake venoms (Gutierrez and Ownby, 2003). These proteins are usually classified into two categories: the Asp49 PLA 2 s, catalytically active, and the Lys49 PLA 2 s, devoid of significant catalytic activity. Nevertheless, Lys49 PLA 2 s induce severe myonecrosis by a non-hydrolytic mechanism (Gutierrez and Ownby, 2003). Current evidence indicates that Lys49 PLA 2 s bind to the plasma membrane of muscle cells and alter their permeability, although a specific receptor has not been identified yet, and their action mechanism is still not well understood (Lomonte et al., 2003). Bothropstoxin-I (BthTX-I) is Lys49 PLA 2 isolated from venom of the South American snake Bothrops jararacussu (Homsi-Brandeburgo et al., 1988). In addi- tion to a potent myotoxic action (Homsi-Brandeburgo ARTICLE IN PRESS www.elsevier.de/etp 0940-2993/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2005.09.004 Ã Corresponding author. Tel.: +55 014 3811 6253; fax: +55 014 3815 3744. E-mail address: gallacci@ibb.unesp.br (M. Gallacci).