Structural and functional characterization of myotoxin, Cr-IV 1, a phospholipase A2 D49 from the venom of the snake Calloselasma rhodostoma.
A new D49 PLA(2) was purified from the venom of Calloselasma rhodostoma after two chromatographic steps. Molecular exclusion chromatography was done through a Protein-Pack 300 SW column (0.78 cm x 30 cm), eluting with 0.25 M ammonium bicarbonate, pH 7.9, at a flow rate of 0.3 ml/min. Reverse-phase HPLC was then performed on mu-Bondapack C-18. The sample was determined to have a molecular mass of 13,870.94 Da MALDI-TOF by mass spectrometry, and the amino acid composition showed that Cr-IV 1 presented a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). Cr-IV 1 presented a sequence of 122 amino acid residues: DLWEFGQMILKETGSLPFPY YTTYGCYCGV GGRGGKPKDA TDRCCFVHDC CYGKLTGCPK TNDRYSYSRL DYTIVCGEGG PCKQICECDK AAAVCFRENL RTYNKKYRYHLKPFCKEPAE TC and a calculated pI value of 8.0. Cr-IV 1 had PLA(2) activity in the presence of a synthetic chromogenic substrate (4-nitro-3-(octanoyloxy)benzoic acid) and showed a rapid cytolytic effect on mouse skeletal muscle myoblasts and myotubes in culture. In mice, Cr-IV 1 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr-IV 1 was determined to be 0.07 mg/k body weight by intracerebroventricular (i.c.v.) injection. The combination of structural and functional information obtained herein classifies Cr-IV 1 as a new member of the D49 PLA(2) family, as it presents the typical behavior of a phospholipase A(2) from this family.
Bonfim
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VL
Department of Biochemistry, Institute of Biology, State University of Campinas, P.O. Box 6109, Zip Code 13083 970, Campinas, SP, Brazil. veralb@yahoo.com
Ponce-Soto
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LA
Martins de Souza
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D
Souza
G H M F
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Baldasso
P A
PA
Eberlin
M N
MN
Marangoni
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D000595:Amino Acid Sequence; D000818:Animals; D001671:Biochemistry / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D054467:Phospholipases A2 / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012911:Snakes; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003435:Crotalid Venoms; C530428:Cr-IV 1 myotoxin, Calloselasma rhodostoma; D054467:Phospholipases A2