Purification and properties of a neutral thiol protease from larval trematode parasite Paragonimus westermani metacercariae.
1. A neutral thiol protease was isolated from the extract of larvae of the mammalian trematode parasite, Paragonimus westermani metacercariae, by arginine-Sepharose, Ultrogel AcA-54 and DEAE-toyopearl column chromatography, measuring its activity by the hydrolysis of Boc-Val-Leu-Lys-MCA as a substrate. 2. The molecular weight of the purified enzyme was estimated to be 22,000 as a single polypeptide by SDS-polyacrylamide gel electrophoresis and was estimated to be 20,000 by size exclusion high-performance liquid chromatography. 3. The activity was suppressed by antipain, E-64, leupeptin, chymostatin, N-tosyl-L-lysine chloromethyl ketone, but was not affected by metallo protease inhibitors or serine protease inhibitors. 4. Studies on the substrate specificity showed that the enzyme hydrolyzed Boc-Val-Leu-Lys-MCA, Z-Phe-Arg-MCA, fluorescein isothiocyanate-labeled collagen, azocoll and casein. 5. The enzyme was found to hydrolyze peptide bonds of oxidized insulin B chain preferentially at the carboxy side of hydrophobic and basic amino acids.
Yamakami
K
K
Hamajima
F
F
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007814:Larva / Q000201:enzymology; D008970:Molecular Weight; D010238:Paragonimus / Q000201:enzymology*; D011480:Protease Inhibitors; D013379:Substrate Specificity
D011480:Protease Inhibitors; D010450:Endopeptidases; D003546:Cysteine Endopeptidases