Purification and characterization of a serine protease (CPM-2) with fibrinolytic activity from the dung beetles.
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 Gln Asp Phe Val Glu Glu Ile Leu. CPM-2 was inactivated by Cu2+ and Zn2+ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and alpha1-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 Aalpha- and Bbeta-chains of fibrinogen and fibrin, and gamma-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.
Ahn
Mi Young
MY
Department of Agricultural Biology, National Institute of Agricultural Science and Technology, Suwon 441-100, Korea. amy@rda.go.kr
Hahn
Bum-Soo
BS
Ryu
Kang Sun
KS
Hwang
Jae Sam
JS
Kim
Yeong Shik
YS
D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D001517:Coleoptera / Q000201:enzymology*; D005338:Fibrin Fibrinogen Degradation Products / Q000032:analysis; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013696:Temperature
D002413:Cations, Divalent; D005338:Fibrin Fibrinogen Degradation Products; D015842:Serine Proteinase Inhibitors; D005340:Fibrinogen; D012697:Serine Endopeptidases