CHARACTERIZATION OF 3 NEUTRAL PROTEASES OF SPIROMETRA-MANSONI PLEROCERCOID

In the pathogenesis of sparganosis, proteases have been considered to play important roles in tissue migration and parasite feeding. Several bands of proteolysis were observed when crude extracts of Spirometra mansoni plerocercoid (sparganum) were examined using gelatin substrate gel at neutral pH, of which two proteases of 198 and 104 kDa were pur ified by two chromatographic steps, and a 36 kDa protease was purified by gelatin-affinity and DEAE-anion exchange chromatography. All the p urified proteases exhibited optimal activity at pH 7.5 and 0.1 M Tris- HCl. Proteolytic activities at 198 and 104 kDa were inhibited specific ally by serine protease inhibitors, and 4-(amidinophenyl)methansulfony l fluoride (APMSF, 0.5 mM) and N-alpha-p-tosyl-L-lysine chloromethyl k etone (TLCK, 1 mM), which strongly suggested that these two proteases were trypsin-like proteases. The activity of the 36 kDa protease was i nhibited by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, 1 mM) a nd chymostatin (0.1 mM), and was potentiated in 10 mM Ca2+ which showe d that the protease had a chymotrypsin-like property. All the protease s were Schiff (PAS) positive. Proteases of 198 and 104 kDa degraded co llagen completely within 24 h. The 36 kDa enzyme cleaved human recombi nant interferon-gamma (rIFN gamma) and bovine myelin basic protein. In addition, all the purified proteins elicited strong antibody response s in the infected patients.