Isolation, characterisation and kinetic studies on SEVA TB ES-31 antigen, a metallo-serine protease of interest in serodiagnosis.

BACKGROUND: SEVA TB Excretory secretory-31 (ES-31) antigen, a glycoprotein isolated from M. tb H37Ra culture filtrate, was found to be useful in the serodiagnosis of pulmonary tuberculosis (TB), extrapulmonary TB and in HIV-TB coinfection. Further, it has been shown to be a zinc containing serine protease. AIM: To isolate and purify SEVA TB ES-31 antigen from M. tb H37Ra culture filtrate and study of its enzyme properties and peptide sequence. METHODS: ES-31 antigen was purified from culture filtrate of M. tuberculosis H37Ra strain by ammonium sulphate precipitation, SDS-PAGE fractionation and FPLC. Protease activity of ES-31 antigen was studied using azocasein as substrate. ES-31 antigen was further fractionated by two dimensional polyacrylamide gel electrophoresis (2D PAGE) followed by LCMS-T analysis. RESULTS: Mycobacterial metallo-serine protease was purified 3096 fold from M. tb H37Ra culture filtrate protein. Purified enzyme showed optimum activity at pH 7.0 at 37 degrees C. Of the four substrates explored, the enzyme has shown maximum activity with azocasein and had a Km value of 0.01 mM with specific activity of 6250 x 10(-6) U/mg protein. Further, analysis of ES-31 antigen by 2D PAGE showed two protein spots (A and B). CONCLUSION: Kinetic studies on SEVA TB ES-31 protein, an immunogen with metallo serine protease activity are reported for the first time. Purified enzyme had a Km value of 0.01 mM with azocasein as substrate. Further, study on structure and biological role of serine protease will be of interest.

Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13 percent of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.

Extracellular serine-proteinases isolated from Streptomyces alboniger: Partial characterization and effect of aprotinin on cellular structure

Streptomyces alboniger ATCC 12461 grown in brain heart infusion (BHI) medium produced two extracellular serine-proteinases, denoted SP I and SP II, which were purified by ammonium sulfate precipitation and aprotinin-agarose affinity chromatography. SP I was purified 88,9-fold and SP II 66,7- fold, with 33.4 percent and 10.4 percent yield, respectively. The optimum pH for the proteinases activity, using a-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate, was 9-10 and the optimum temperature was 37ºC. The proteolytic activity of SP I and SP II was inhibited by aprotinin and SP I was partially inhibited by leupeptin, both serine-proteinase inhibitors. S. alboniger growth in BHI-liquid medium decreased when 5 mg/ml, 10 mg/ml of aprotinin was used, being completely inhibited with 20 mg/ml and 40 mg/ml. At the ultrastructural level, aprotinin-treated S. alboniger cells showed swelling of the bacterial body and condensation of the genetic material, probably related to the inhibition of its growth

Serine proteinase from rice bean.

A trypsin like serine-proteinase of M(r) 16,000 Da, optimally active at pH 8.4 on N-benzoyl-arginine ethyl ester (BAEE) was purified from 4-day old germinated seeds of rice bean, Vigna umbellata (Thunb), by ammonium sulphate precipitation, gel filtration, ion-exchange chromatography and by high performance liquid chromatography (HPLC). The purity of the enzyme was checked by polyacrylamide gel electrophoresis (PAGE). The enzyme activity was studied on natural substrates like casein, haemoglobin and vicilin, a rice bean storage protein. The activity of the enzyme was completely inhibited by phenylmethylsulfonyl fluoride, but not by iodoacetamide and HgCl2, suggesting it to be a serine protease. Loss of activity in presence of EDTA was reversed by addition of Ca2+.