Purification, primary structures and evolution of coagulant proteases from Deinagkistrodon actus venom.
Deinagkistrodon (formerly Agkistrodon) actus (Taiwan) snake venom was found to contain at least seven closely related coagulant proteases. One of them, named actibin, was purified to homogeneity by means of four chromatographic steps. Actibin acted on fibrinogen to form fibrin clots with extremely high specific activity of 1,630 NIH units/mg and preferentially released fibrinopeptide A. Actibin was an acidic glycoprotein (pI 3.4) with molecular weight of 41,000, which was reduced to 28,800 after deglycosylation with N-glycanase. The k(cat)/K(m) values of actibin for hydrolysis of tosyl-l-arginine methyl ester and benzoyl-l-arginine p-nitroanilide were one-third to a half those for thrombin, reflecting a high potency of actibin in fibrinogen clotting. The amidase activities of actibin and its family proteases were inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, indicating that actibin and its family proteases are serine proteases. Four cDNAs, named DaP1 and DaP7-DaP9, encoding D. actus coagulant proteases were cloned. All cDNAs contain an open reading frame of 780 bp coding for 260 amino acid residues, including a signal peptide of 24 amino acid residues. Their amino acid sequences predicted are highly homologous to one another with one to five amino acid substitutions. When four D. actus protease cDNAs were compared with the cDNAs coding for Trimeresurus flavoviridis and T. gramineus venom serine proteases, accelerated evolution was clearly observed. Similarity of the nucleotide sequences of four D. actus protease cDNAs with no synonymous and one to five nonsynonymous substitutions seems not to be in direct conformity with accelerated evolution. This possibly suggests that they have evolved to a similar direction to enhance their clotting activity rather than to produce other physiological activities.
Nikandrov
Nikolai N
NN
Laboratory of Chemistry and Technology of Animal Products, Division of Biological Resources and Environmental Science, Graduate School of Kyushu University, Fukuoka 812-8581, Japan.
Deshimaru
Masanobu
M
Tani
Ayako
A
Chijiwa
Takahito
T
Shibata
Hiroki
H
Chang
Chang-Chun
CC
Fukumaki
Yasuyuki
Y
Ito
Tatsumi
T
Ohno
Motonori
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D003001:Cloning, Molecular; D016000:Cluster Analysis; D003029:Coagulants / Q000302:isolation & purification* / Q000378:metabolism; D003435:Crotalid Venoms / Q000737:chemistry*; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D005340:Fibrinogen / Q000378:metabolism; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014106:Tosylarginine Methyl Ester / Q000378:metabolism; D017819:Viperidae
D003029:Coagulants; D003435:Crotalid Venoms; D018076:DNA, Complementary; D005340:Fibrinogen; D014106:Tosylarginine Methyl Ester; D001586:Benzoylarginine Nitroanilide; D010447:Peptide Hydrolases