Primary sequence determination of the most basic myonecrotic phospholipase A2 from the venom of Vipera russelli.
The most basic phospholipase A2 (PLA2), VRV-PL-VIIIa, was purified from (Sri Lankan) Vipera russelli venom. It is a major component of the venom, contributing over 40% to the whole venom PLA2 activity. The purity of VRV-PL-VIIIa was ascertained by electrophoresis and by reverse phase high-pressure liquid-chromatography (RP-HPLC). VRV-PL-VIIIa had an apparent mol. wt of 13,000 and was a single polypeptide. The protein was reduced, pyridylethylated and subjected to sequence analysis. The N-terminal amino acid sequence was established up to the 39th residue. Pyridylethylated VRV-PL-VIIIa was digested with endoprotease Glu-C, and several peptides were purified by RP-HPLC; six purified peptides were sequenced. The sequence of the C-terminal was established by sequencing a CNBr-produced peptide purified by RP-HPLC. Several peptides were also generated by digestion with endoprotease Asp-N. Two peptides were sequenced to obtain overlapping regions. The complete structure was deduced from sequences of overlapping peptides and through homology with other group II PLA2 sequences. Sequence homology was greatest with ammodytoxin A: 99 amino acid residues out of 121 occurred in identical positions. Myotoxin III of Bothrops asper showed 73% homology, 89 out of 121 residues. In agreement with the sequence data, polyclonal antiserum against VRV-PL-VIIIa cross-reacted in ELISA with ammodytoxin A and, to a lesser extent, with caudoxin.
Gowda
V T
VT
Toxinology Division, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21701-5011.
Schmidt
J
J
Middlebrook
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D003439:Crotoxin / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D054501:Group II Phospholipases A2; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D054467:Phospholipases A2; D030162:Reptilian Proteins; D017840:Daboia; D017386:Sequence Homology, Amino Acid; D014757:Viper Venoms / Q000737:chemistry / Q000201:enzymology*
D030162:Reptilian Proteins; D014757:Viper Venoms; D003439:Crotoxin; C068117:ammodytoxin A; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; C034914:caudoxin