A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms.
As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.
Choi
M H
MH
Department of Parasitology, SNUMRC, Seoul National University College of Medicine, Seoul, Korea. mhchoi@plaza.snu.ac.kr
Choe
S C
SC
Lee
S H
SH
D000818:Animals; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000502:physiology; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D006790:Host-Parasite Interactions; D006801:Humans; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D012911:Snakes; D014200:Trematoda / Q000201:enzymology* / Q000472:pathogenicity
D016326:Extracellular Matrix Proteins; D003546:Cysteine Endopeptidases