Purification and characterization of a serine protease with fibrinolytic activity from the dung beetles, Catharsius molossus.
Catharsius protease-1 (CPM-1) was isolated from the whole body of the dung beetles, Catharsius molossus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel Blue gel). The purified CPM-1 that has a molecular weight of 27 kDa was assessed homogeneous by SDS-polyacrylamide gel electrophoresis and an isoelectric point of 4.4 was determined by isoelectric focusing. N-terminal amino acid sequence of the protease was composed of Ile-Val-Gly-Gly-Gln-Ala-Val-Glu-Ile-Gly-Asp-Tyr-Pro-Ala-Gln. The enzyme 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-antitrypsin. However, EDTA, EGTA, cysteine, beta-mercaptoethanol, E64, chymostatin, elastatinal and TPCK did not/less affect activity. Also, antiplasmin and antithrombin III were not sensitive to CPM-1. On the basis of amidolytic activity test, CPM-1 preferably hydrolysed chromogenic protease substrates containing Arg or Lys residues of the P1 position at pH 7.0 and 37 degrees C. CPM-1 preferentially cleaved the oxidized B-chain of insulin between Arg(22) and Gly(23). CPM-1 readily digested Aalpha- and gamma-chains and more slowly Bbeta-chain of fibrinogen. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. D-dimer concentration increased on incubation of cross-linked fibrin with CPM-1, indicating that the enzyme has a significant fibrinolytic activity.
Ahn
Mi Young
MY
Department of Sericulture and Entomology, National Institute of Agricultural Science and Technology, 61 Sudun-Dong, Kwonsun-gu, Suwon 441-100, South Korea. amy@rda.go.kr
Hahn
Bum-Soo
BS
Ryu
Kang Sun
KS
Kim
Jin Won
JW
Kim
Iksoo
I
Kim
Yeong Shik
YS
D000595:Amino Acid Sequence; D000818:Animals; D001517:Coleoptera; D005338:Fibrin Fibrinogen Degradation Products / Q000032:analysis; D005340:Fibrinogen / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects*; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007526:Isoelectric Point; D019216:Metals, Heavy / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D005338:Fibrin Fibrinogen Degradation Products; D007328:Insulin; D019216:Metals, Heavy; D015842:Serine Proteinase Inhibitors; C036309:fibrin fragment D; D005340:Fibrinogen; D012697:Serine Endopeptidases