Structural and functional properties of BaTX, a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Bothrops alternatus.
BaTX PLA(2), a K49 phospholipase A(2) homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on mu-Bondapack C-18. A molecular mass of 13898.71 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that BaTX has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). The complete amino acid sequence of BaTX PLA(2) contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA(2)s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA(2)s. The sequence was SLFELGKMIL QETGKNPAKS YGAYYCYCGW GGQGQPKDAT DRCCYVHKCC YKKLTGCNPK KDRYSYSWKD KTIVCGENNS CLKELCECDK AVAICLRENL NTYNKKYRYY LKPLCKKADA C. In mice, BaTX induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. The LD(50) of BaTX was 7 mug/g body weight, by intravenous route. In vitro, the toxin caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparations. The blockage 50% was achieved at a concentration of 0.03 microM: 40+/-0.4 min and 0.07 microM: 35+/-0.3 min. Moreover, this protein induced a rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. Thus, the combined structural and functional information obtained identify BaTX as a new member of the K49 PLA(2) family, which presents the typical bioactivities described for such proteins.
Ponce-Soto
Luis Alberto
LA
Biochemistry Department, Institute of Biology, Medical Sciences Faculty, State University of Campinas (UNICAMP), Campinas, SP, Brazil. poncesoto@yahoo.com.ar
Lomonte
Bruno
B
Gutiérrez
José María
JM
Rodrigues-Simioni
Lea
L
Novello
José Camillo
JC
Marangoni
Sergio
S
D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002645:Chickens; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004487:Edema / Q000139:chemically induced; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry; D007928:Lethal Dose 50; D008239:Lysine; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000187:drug effects / Q000473:pathology; D032448:Myoblasts, Skeletal / Q000187:drug effects; D009336:Necrosis; D009469:Neuromuscular Junction / Q000187:drug effects; D010741:Phospholipases A / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity*; D054467:Phospholipases A2; D011487:Protein Conformation; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D003435:Crotalid Venoms; D007527:Isoenzymes; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine