Purification and amino acid sequence of brevilysin L6, a non-hemorrhagic metalloprotease from Agkistrodon halys brevicaudus venom.
A non-hemorrhagic proteinase, brevilysin L6 (L6), has been purified to homogeneity from Agkistrodon halys brevicaudus venom by gel filtration and DEAE-Toyopearl 650M chromatography. It is an acidic protein with an isoelectric point of 4.8, and its molecular mass was estimated to be 21.5 kDa by SDS-PAGE. The optimum pH of L6 was about 9. EDTA and o-phenanthroline inhibited the proteolytic activity, suggesting that L6 is a metalloprotease. Cysteine also inhibited the activity of L6, but glutathione did not. The protein was stable in the pH range of 5-8.5 and below 40 degreesC. Calcium ions had no effect on the proteolytic activity of L6 or on its thermal stability. The enzyme preferentially cleaved X-Leu, X-Phe, X-Val, and X-His bonds. L6 showed weak alpha-fibrinogenase activity. The complete amino acid sequence of L6 was also determined by manual Edman degradation. L6 is a non-glycosylated single-chain polypeptide consisting of 203 residues with an N-terminal pyroglutamic acid and a calculated molecular weight of 22,713 Da. Its entire sequence is highly homologous to those of other metalloproteases from various snake venoms. A zinc-binding motif, HEXXHXXGXXH, is located at residues 143-153 in the sequence of L6.
Terada
S
S
Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka, 814-0180, Japan.
Hori
J
J
Fujimura
S
S
Kimoto
E
E
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D003435:Crotalid Venoms / Q000201:enzymology*; D004795:Enzyme Stability; D005343:Fibrinolytic Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014757:Viper Venoms / Q000302:isolation & purification*
D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D014757:Viper Venoms; D008666:Metalloendopeptidases; C118463:brevilysin L6