Archives of Pharmacal Research

  • 제28권7호
  • /
  • Pages.816-822
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  • 2005
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  • 0253-6269(pISSN)
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  • 1976-3786(eISSN)
대한약학회 (The Pharmaceutical Society of Korea)
권호 표지

Purification and Characterization of a Serine Protease (CPM-2) with Fibrinolytic Activity from the Dung Beetles

  • Ahn, Mi-Young (Department of Agricultural Biology, National Institute of Agricultural Science and Technology) ;
  • Hahn, Bum-Soo (Plant Metabolite Engineering Team, National Institute of Agricultural Biotechnology) ;
  • Ryu, Kang-Sun (Department of Agricultural Biology, National Institute of Agricultural Science and Technology) ;
  • Hwang, Jae-Sam (Department of Agricultural Biology, National Institute of Agricultural Science and Technology) ;
  • Kim, Yeong-Shik (Natural Products Research Institute, Seoul National University)
  • 발행 : 2005.07.01
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초록

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

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