title: Purification and characterization of a prothrombin-activating protease from Nephila clavata.
abstract: We report upon the purification and characterization of a novel prothrombin-activating enzyme from the body fluid (total homogenates of isolated digestive tract without eggs, spinnerets and silk glands) of the spider, Nephila clavata by a combination of acetone fractionation, ion exchange, and Soybean trypsin inhibitor-Sepharose chromatography. Analysis of the purified enzyme with SDS-PAGE and gel filtration revealed a single polypeptide chain with an apparent molecular weight of 24kDa. The proteolytic activity of the enzyme was stable up to 50 degrees C, however, it became unstable over 55 degrees C. The enzyme had an optimum pH of 8, and Ca(2+) was not required for the enzyme activity. According to inhibition profiles obtained with several serine protease inhibitors such as PMSF and benzamidine, the purified protease is a member of the serine proteases. Bz-Ile-Glu(gamma-OR)- Gly-Arg-pNA and Z-Arg-Gly-Arg-pNA which are known as substrates for factor Xa, were hydrolyzed favorably by the enzyme. And the Nephila protease could produce thrombin from prothrombin at nM range, and form the turbid ring using fibrinogen-agarose plate. The results obtained confirmed that the purified protease is a potent prothrombin-activating activity belonging to the family of serine protease.
authors.affiliation: Institute of Medical Science, Inha University, Chung-Ku, Inchon 400-103, South Korea.
authors.forename: Han-Seung
authors.initials: HS
authors.lastname: Joo
authors.forename: Gun-Chun
authors.initials: GC
authors.lastname: Park
authors.forename: Woo Ri
authors.initials: WR
authors.lastname: Cho
authors.forename: Eunsik
authors.initials: E
authors.lastname: Tak
authors.forename: Seung R
authors.initials: SR
authors.lastname: Paik
authors.forename: Chung-Soon
authors.initials: CS
authors.lastname: Chang
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D011506:Proteins / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013112:Spiders / Q000737:chemistry*; D013917:Thrombin / Q000378:metabolism*
chemical_list: D011506:Proteins; D011516:Prothrombin; D012697:Serine Endopeptidases; D013917:Thrombin