Neurotoxic, Myotoxic and Cytolytic Activities of the New Basic PLA 2 Isoforms BmjeTX-I and BmjeTX-II Isolated from the Bothrops marajoensis (Marajo ´ Lancehead) Snake Venom L. A. Ponce-Soto • D. Martins-de-Souza • S. Marangoni Published online: 27 February 2010 Ó Springer Science+Business Media, LLC 2010 Abstract The BmjeTX-I and BmjeTX-II isoforms of PLA 2 were purified from Bothrops marajoensis venom by ion-exchange chromatography and reverse phase HPLC. Both isoforms showed a molecular mass of 13808.89 Da (BmjeTX-I) and 13863.97 Da (BmjeTX-II) determined by based on the determined primary structures and SDS– PAGE and confirmed experimentally by MALDI-TOF mass spectrometry. Multiple alignment of BmjeTX-I and BmjeTX-II isoforms of PLA 2 show high degree of homology with basic PLA 2 myotoxins from other Bothrops venoms. Ex vivo, both isoforms caused a blockade of the neuromuscular transmission in young chick biventer cer- vicis preparations in a similar way to other Bothrops spe- cies. In chick preparations, contractures to exogenous acetylcholine (55 and 110 lM) or KCl (13.4 mM) were unaltered after complete blockade for the both isoforms BmjeTX-I and BmjeTX-II of PLA 2 . These results, which strongly suggested a presynaptic mechanism of action for these toxins. In mice, both isoforms induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Both isoforms BmjeTX-I and BmjeTX-II of PLA 2 also induced moderate marked paw edema, evi- dencing the local increase in vascular permeability. Since both isoforms of PLA 2 exert a strong proinflammatory effect, the enzymatic hydrolysis of phospholipids might be relevant for this phenomenon and produced cytotoxicity in murine skeletal muscle C2C12 myoblasts and myotubes. Keywords Phospholipase A 2 Á Snake venom Á Bothrops marajoensis Á Myotoxin Á Neurotoxin Á Cytotoxitic Á C2C12 Á Isoforms 1 Introduction Secreted phospholipases A 2 (PLA 2 ) constitute a distinct group of enzymes that are abundant in animal venoms. Snake venom PLA 2 s display varied pharmacological properties which include myotoxicity [13, 14, 21], edema formation [30], presynaptic [36] plus post-synaptic neuro- toxicity [3], cardiotoxicity [10] and platelet aggregation [20]. One of the major problems in understanding the mech- anism of b-neurotoxicity at the molecular level is the site(s) to which b-neurotoxins localize to produce toxic effects. One view is that b-neurotoxins act on the external side of the plasma membrane; another is that they are endocyto- sed, acting also on intracellular membranes and binding to certain targets within the nerve terminal [39]. The b-Neurotoxicity is a process that depends essen- tially on phospholipase activity of a toxin, but many years ago it became evident that this is not a property that could explain the difference in pharmacological action of neu- rotoxic and non-neurotoxic sPLA 2 s. b-Neurotoxicity could not be related to enzymatic specificity of sPLA 2 s or to their enzymatic potency. Secreted PLA 2 s are enzymes that bind to biological membranes and hydrolyze them, as well as presynaptic membranes. For example, using Atx, from L. A. Ponce-Soto (&) Á D. Martins-de-Souza Á S. Marangoni Department of Biochemistry, Institute of Biology (IB), State University of Campinas (UNICAMP), P.O. Box 6109, Zip code 13083-970 Campinas, SP, Brazil e-mail: poncesoto@yahoo.com.ar L. A. Ponce-Soto Department of Pharmacology, Faculty of Medical Science (FCM), State University of Campinas (UNICAMP), Campinas, SP, Brazil D. Martins-de-Souza Max Planck Institute of Psychiatry, Munich, Germany 123 Protein J (2010) 29:103–113 DOI 10.1007/s10930-010-9229-5