title: Purification, characterization and gene cloning of Da-36, a novel serine protease from Deinagkistrodon acutus venom.
abstract: A serine protease termed Da-36 was isolated from crude venom of Deinagkistrodon acutus. The enzyme was a single chain protein with an apparent molecular weight of 36,000 on SDS-PAGE with an isoelectric point of 6.59. Da-36 could clot human plasma by cleaving the Aα, Bβ and γ chains of fibrinogen and also exhibited arginine esterase activity. The proteolytic activity of Da-36 toward TAME was strongly inhibited by PMSF and moderately affected by benzamidine and aprotinin, indicating that it was a serine protease. Meanwhile, Da-36 showed stability with wide temperature (20-50 °C) and pH value ranges (pH 6-10). Divalent metal ions of Ca(2+), Mg(2+), and Mn(2+) had no effects but Zn(2+) and Cu(2+) inhibited the arginine esterase activity of Da-36. Total DNA was extracted directly from the lyophilized crude venom and the gene (5.5 kbp) coding for Da-36 had been successfully cloned. Sequence analysis revealed that the Da-36 gene contained five exons and four introns. The mature Da-36 was encoded by four separate exons. The deduced mature amino acid sequence of Da-36 was in good agreement with the determined N-terminal sequence of the purified protein and shared high homology with other serine proteases isolated from different snake venoms. Blast search using amino acid sequence of Da-36 against public database revealed that Da-36 showed a maximal identity of 90% with both Dav-X (Swiss-Prot: Q9I8W9.1) and thrombin-like protein 1 (GenBank: AAW56608.1) from the same snake species, indicating that Da-36 is a novel serine protease.
authors: Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China.
authors: Ying
authors: Y
authors: Zheng
authors: Feng-Ping
authors: FP
authors: Ye
authors: Jie
authors: J
authors: Wang
authors: Guo-Yang
authors: GY
authors: Liao
authors: Yun
authors: Y
authors: Zhang
authors: Quan-Shui
authors: QS
authors: Fan
authors: Wen-Hui
authors: WH
authors: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000201:enzymology*; D017839:Crotalus / Q000502:physiology*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D003435:Crotalid Venoms; D005340:Fibrinogen; C019724:arginine esterase; D002265:Carboxylic Ester Hydrolases; D057057:Serine Proteases