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Purification and characterization of a plasmin-like protease from Tenodera sinensis (Chinese mantis)
1Natural Products Research Institute, Seoul National University, 28 Yeonkun-Dong, Jongno-Ku, Seoul 110-460, South Korea.
Abstract:
A novel type of protease (mantis egg fibrinolytic enzyme, MEF-2) was isolated from the egg cases of Tenodera sinensis. The protease was homogeneous by SDS-PAGE and its apparent molecular mass was 32,900 Da. The amino acids in the N-terminal region were Ile-Val-Gly-Gly-Glu-Glu-Ala-Val-Ala-Gly-Asp-Phe-Pro-Ile-Val-Ser-Leu-Gln-Glu. The enzyme was inhibited by PMSF, TLCK, aprotinin, benzamidine, soybean trypsin inhibitor and also slightly by elastatinal, EDTA, EGTA, cysteine and beta-mercaptoethanol, but TPCK, iodoacetate and E-64 did not affect the activity. MEF-2 was not sensitive to alpha(1)-antitrypsin but antithrombin III and alpha(2)-antiplasmin inhibited the enzyme. MEF-2 preferentially cleaved the oxidized B-chain of insulin between Arg(22) and Gly(23). Among chromogenic protease substrates, the most susceptible to MEF-2 hydrolysis was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at 30 degrees C and pH 5.0. These results indicate that MEF-2 belongs to the trypsin family. Upon incubation of crosslinked fibrin with MEF-2, a steady increase of D-dimer suggests that the enzyme has a strong fibrinolytic activity. In conclusion, MEF-2 is a new type of proteolytic enzyme and has some potential for practical application in fibrinolysis.
Insights
A novel protease, mantis egg fibrinolytic enzyme (MEF-2), was isolated from praying mantis egg cases. This enzyme exhibits strong fibrinolytic activity, suggesting potential applications in treating blood clots.
Area of Science:
- Biochemistry
- Enzymology
- Proteomics
Background:
- Proteases play crucial roles in biological processes.
- Fibrinolytic enzymes are vital for dissolving blood clots.
- Novel enzymes offer potential for therapeutic advancements.
Purpose of the Study:
- To isolate and characterize a novel protease from Tenodera sinensis egg cases.
- To investigate the enzymatic properties and fibrinolytic activity of the isolated protease, designated MEF-2.
- To determine the potential applications of MEF-2 in fibrinolysis.
Main Methods:
- Isolation and purification of protease using SDS-PAGE.
- N-terminal amino acid sequencing.
- Enzyme inhibition assays with various inhibitors (PMSF, TLCK, aprotinin, etc.).
- Substrate specificity analysis using insulin B-chain and chromogenic substrates.
- Fibrinolytic activity assay by monitoring D-dimer formation.
Main Results:
- A novel protease, MEF-2, with a molecular mass of 32,900 Da was isolated.
- MEF-2 demonstrated inhibition by PMSF, aprotinin, and benzamidine, suggesting serine protease activity.
- The enzyme preferentially cleaved insulin B-chain and showed maximal activity at pH 5.0 and 30°C.
- MEF-2 exhibited significant fibrinolytic activity, indicated by increased D-dimer formation.
Conclusions:
- MEF-2 is a novel serine protease belonging to the trypsin family.
- The enzyme possesses potent fibrinolytic properties.
- MEF-2 shows promise for practical applications in fibrinolysis and thrombosis treatment.