caa a22 4500 397557647 CHVBK 20180308164807.0 cr unu---uuuuu 161202e199605 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021319 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021319 Purification and Molecular Cloning of Calobin,a Thrombin-Like Enzyme from Agkistrodon caliginosus (Korean Viper) [Elektronische Daten] [Bum-Soo Hahn, Kyoung-Youl Yang, Eun-Mi Park, Moo Chang, Yeong-Shik Kim] A thrombin-like enzyme, calobin, has been purified to homogeneity from the venom of Agkistrodon caliginosus by a procedure involving Bio-Gel P-100, Mono S, and Pro-RPC. The enzyme was identified as a monomer with a molecular weight of 34,000 on SDS-PAGE, and its isoelectric point was 6.2. Calobin acted on fibrinogen to form fibrin with a specific activity of 226 NIH equivalent units, and also exhibited arginine esterase activity. The enzyme predominantly cleaved the α-chain of fibrinogen with little degradation of the β-chain.It contained abundant asparagine/aspartic acid residues, but very few tyrosine or methionine residues. The proteolytic activity of the enzyme with TAME as a substrate was higher than that of thrombin. However, it showed neither lysine esterase nor caseinolytic activity. The enzyme activity was strongly inhibited by PMSF, and moderately by benzamidine and soybean trypsin inhibitor, indicating it is a serine protease. On the other hand, the enzyme activity was not inhibited by hirudin or aprotinin. cDNA (1.6kb) for calobin has been cloned from an A.caliginosus; cDNA library. The cDNA sequence indicates that calobin is synthesized as a pre-zymogen of 262 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acid residues. The cDNA sequence encodes a 238-amino acid residue molecule exhibiting strong amino acid sequence homology to those of ancrod, batroxobin, and flavoxobin isolated from other snake venoms. Calobin contains 12 cysteine residues. As judged on alignment of the amino acid sequences of other thrombin-like enzymes (batroxobin, ancrod, and flavoxobin), calobin constitute the formation of six disulfide bridges. Amino acid residues, His43,Asp88,and Ser182, which are thought to be the catalytic active site are highly conserved. As calobin is a glycoprotein, its possible glycosylation site, Asn-X-Thr, is located at amino acid residues 81-83. © by the Journal of Biochemistry Regular Papers nationallicence Agkistrodon caliginosus nationallicence cDNA sequence nationallicence purification nationallicence thrombin-like enzyme nationallicence Hahn Bum-Soo Natural Products Research Institute, Seoul National University Seoul 110-460, Korea aut Yang Kyoung-Youl Natural Products Research Institute, Seoul National University Seoul 110-460, Korea aut Park Eun-Mi Department of Chemistry, University of Incheon Incheon 402- 749, Korea aut Chang Moo Natural Products Research Institute, Seoul National University Seoul 110-460, Korea aut Kim Yeong-Shik Natural Products Research Institute, Seoul National University Seoul 110-460, Korea aut The Journal of Biochemistry Oxford University Press 119/5(1996-05), 835-843 0021-924X 119:5<835 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021319 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021319 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hahn Bum-Soo Natural Products Research Institute, Seoul National University Seoul 110-460, Korea aut NATIONALLICENCE 700 1- Yang Kyoung-Youl Natural Products Research Institute, Seoul National University Seoul 110-460, Korea aut NATIONALLICENCE 700 1- Park Eun-Mi Department of Chemistry, University of Incheon Incheon 402- 749, Korea aut NATIONALLICENCE 700 1- Chang Moo Natural Products Research Institute, Seoul National University Seoul 110-460, Korea aut NATIONALLICENCE 700 1- Kim Yeong-Shik Natural Products Research Institute, Seoul National University Seoul 110-460, Korea aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/5(1996-05), 835-843 0021-924X 119:5<835 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford