Purification, cDNA cloning and molecular characteristic of a fibrinolytic enzyme from the venom of Agkistrodon acutus.
A nonglycoprotein-like fibrinolytic enzyme ((FIB-I) was purified from the crude venom of Agkistrodon acutus by CM-Sepharose CL-6B and DEAE-Sepharose CL-6B ion exchange chromatography and then by FPLC through Superose 12 gel filtration. Its molecular weight is about 23 kDa and isoelectric point is near 6.0. It not only has fibrinolytic and caseinolytic activity, but also can hydrolyze BAEE. The local hemorrhagic activity was found in mice after the subcutaneous injection of this enzyme. EDTA can inhibit its fibrinolytic activity completely, but PMSF and arrowhead proteinase inhibitor have no such obvious inhibitory effect, thus implying that FIB-I is a metalloproteinase. The N-terminal ten amino acid residues 'STEFQRYMEI' of FIB-I was elucidated. A full-length cDNA gene of this enzyme was cloned by using RT-PCR from the total RNA extracted from the snake venom gland and FIB-I was expressed in E. coli. Having analyzed the sequence, we found that it had a typical zinc-chelating characteristic as 'HEXXHXXGXXHD.'
Du
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Shanghai Institute of Biochemistry, Academia Sinica, China.
Pan
H
H
Jin
Y
Y
Zhu
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H
Wu
X F
XF
Zhou
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YC
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000201:enzymology*; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006470:Hemorrhage / Q000139:chemically induced; D008297:Male; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D003435:Crotalid Venoms; D018076:DNA, Complementary; D001120:Arginine; C009727:benzoylarginine ethyl ester; C114433:FIB-I enzyme; D008666:Metalloendopeptidases