Characterization of three 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 purified by two chromatographic steps, and a 36 kDa protease was purified by gelatin-affinity and DEAE-anion exchange chromatography. All the purified proteases exhibited optimal activity at pH 7.5 and 0.1 M Tris-HCl. Proteolytic activities at 198 and 104 kDa were inhibited specifically by serine protease inhibitors and 4-(amidinophenyl)methanesulfonyl fluoride (APMSF, 0.5 mM) and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK, 1 mM), which strongly suggested that these two proteases were trypsin-like proteases. The activity of the 36 kDa protease was inhibited by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, 1 mM) and chymostatin (0.1 mM), and was potentiated in 10 mM Ca2+ which showed that the protease had a chymotrypsin-like property. All the proteases were Schiff (PAS) positive. Proteases of 198 and 104 kDa degraded collagen completely within 24 h. The 36 kDa enzyme cleaved human recombinant interferon-gamma (rIFN gamma) and bovine myelin basic protein. In addition, all the purified proteins elicited strong antibody responses in the infected patients.
Kong
Y
Y
Department of Parasitology, College of Medicine, Chung-Ang University, Seoul, Korea.
Chung
Y B
YB
Cho
S Y
SY
Choi
S H
SH
Kang
S Y
SY
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005780:Gelatin / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000201:enzymology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D012911:Snakes / Q000469:parasitology; D013031:Sparganosis / Q000469:parasitology*; D013146:Spirometra / Q000201:enzymology*
D006454:Hemoglobins; D011480:Protease Inhibitors; D005780:Gelatin; D010450:Endopeptidases