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Title
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Characterization of multiple metalloproteinases with fibrinogenolytic activity from the venom of Taiwan habu (Trimeresurus mucrosquamatus): protein microsequencing coupled with cDNA sequence analysis.
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Authors
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K.F.Huang,
C.C.Hung,
F.M.Pan,
L.P.Chow,
A.Tsugita,
S.H.Chiou.
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Ref.
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Biochem Biophys Res Commun, 1995,
216,
223-233.
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PubMed id
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Abstract
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Three fibrinogenolytic proteases were isolated and purified from the venom of
Taiwan habu (Trimeresurus mucrosquamatus) using anion-exchange and
gel-filtration chromatographies followed by cation-exchange HPLC. Further
characterization of these purified fractions with fibrinogenase activity
indicated that they are single-chain proteases of approximately 24 kDa,
possessing strong cleaving activity mainly on the A alpha and less on B beta and
gamma chains of fibrinogen subunit chains. Enzyme activities were strongly
inhibited by EDTA or 1,10-phenanthroline and not by phenylmethanesulfonyl
fluoride, suggesting that these fibrinogenases belong to the family of
metalloproteinases and not thrombin-like serine proteases. N-Terminal sequence
analysis of these proteases failed to show any free amino-terminal residues,
thus hampering the sequence determination by conventional sequencing strategy.
Microsequencing on the electroblotted fragments of CNBr-treated proteases
separated on SDS-PAGE was then used to determine the partial sequences. Sequence
comparison of the determined partial sequences of these proteins with published
sequences of the protein data bank revealed that they showed sequence homology
with H2-protease. HR2a and protrigramin, which were all shown to belong to
metalloproteinases present in various snake venoms. Polymerase chain reaction
(PCR) was employed to amplify cDNAs constructed from the poly(A)+RNA of fresh
venom glands of the same snake species to facilitate cloning and sequencing of
these proteases. Sequencing several positive clones containing amplified cDNAs
revealed the existence of one fibrinogenase in the Taiwan habu, which was
contained within one complete cDNA encoding the preproproteinase precursor of
hemorrhagic metalloproteinases.
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