Purification and characterization of a serine protease with fibrinolytic activity from Tenodera sinensis (praying mantis)

Mantis egg fibrolase (MEF) was purified from the egg cases of Tenodera sine nsis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60 a nd affinity chromatography on DEAE Affi-Gel blue gel. The protease was asse ssed homogeneous by SDS-polyacrylamide gel electrophoresis and has a molecu lar mass of 31 500 Da. An isoelectric point of 6.1 was determined by isoele ctric focusing. Amino acid sequencing of the N-terminal region established a primary structure composed of Ala-Asp-Val-Val-Gln-Gly-Asp-Ala-Pro-Ser. ME F readily digested the A alpha- and B beta-chains of fibrinogen and more sl owly the gamma-chain. The nonspecific action of the enzyme results in exten sive hydrolysis of fibrinogen and fibrin releasing a variety of fibrinopept ide. The enzyme is inactivated by Cu2+ and Zn2+ and inhibited by PMSF and c hymostatin, yet elastinal, aprotinin, TLCK, TPCK, EDTA, EGTA, cysteine, bet a-mercaptoethanol, iodoacetate, E64, benzamidine and soybean trypsin inhibi tor do not affect activity. Antiplasmin was not sensitive to MEF but antith rombin III inhibited the enzymatic activity of MEF, Among chromogenic prote ase substrates, the most sensitive to MEF hydrolysis was benzoyl-Phe-Val-Ar g-p-nitroanilide with maximal activity at pH 7.0 and 30 degrees C, MEF pref erentially cleaved the oxidized B-chain of insulin between Leu(15) and Tyr( 16). D-Dimer concentrations increased on incubation of cross-linked fibrin with MEF, indicating the enzyme has a strong fibrinolytic activity. (C) 199 9 Elsevier Science B.V. All rights reserved.