title: Biochemical and pharmacological characterization of PhTX-I a new myotoxic phospholipase A2 isolated from Porthidium hyoprora snake venom.
abstract: This paper reports the biochemical and pharmacological characterization of a new myotoxic PLA(2) (EC 3.1.1.4) called PhTX-I, purified from Porthidium hyoprora venom by one step analytical chromatography reverse phase HPLC. The homogeneity of the PhTX-I fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14.249Da and constituted of a single polipeptidic chain. Amino acid sequence was determined by "de novo sequencing," in tandem mass spectrometry, belonging to D49-PLA(2) enzyme class and exhibiting high identity (44-90%) with other myotoxics PLA(2) from snake venoms. The enzymatic investigation showed maximal activity at pH 8 and 35-45°C. This activity was dependent on Ca(2+), other cations (Mg(2+), Mn(2+), Cd(2+) and Zn(2+)) reduced notably the enzymatic activity, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca(2+). Ex vivo, whole venom and PhTX-I PLA(2) caused blockade of the neuromuscular transmission in young chick biventer cervicis preparations similar to other isolated snake venom toxins from the Bothrops genus. In vivo, both induced local myotoxicity and systemic interleukin-6 response upon intramuscular injection, additionally, induced moderate footpad edema. In vitro, both induced low cytotoxicity in skeletal muscle myoblasts, however PhTX-I PLA(2) was able to lyse myotubes.
authors.affiliation: Department of Biochemistry, Institute of Biology, State University of Campinas, SP, Brazil.
authors.forename: Salomón
authors.initials: S
authors.lastname: Huancahuire-Vega
authors.forename: Luis Alberto
authors.initials: LA
authors.lastname: Ponce-Soto
authors.forename: Daniel
authors.initials: D
authors.lastname: Martins-de-Souza
authors.forename: Sergio
authors.initials: S
authors.lastname: Marangoni
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology* / Q000633:toxicity*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000473:pathology; D032448:Myoblasts, Skeletal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009469:Neuromuscular Junction / Q000187:drug effects; D054467:Phospholipases A2 / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D003435:Crotalid Venoms; D007770:L-Lactate Dehydrogenase; D054467:Phospholipases A2