Purification and characterization of a cathepsin L-like enzyme from the body wall of the sea cucumber Stichopus japonicus.
Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS-PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine7-amido-4-methylcoumarin with K(m) (69.92 microM) and k(cat) (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 degrees C. It showed thermal stability below 40 degrees C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.
Zhu
Bei-Wei
BW
College of Bio and Food Technology, Dalian Polytechnic University, PR China. zhubeiwei@163.com
Zhao
Lu-Lu
LL
Sun
Li-Ming
LM
Li
Dong-Mei
DM
Murata
Yoshiyuki
Y
Yu
Lei
L
Zhang
Lei
L
D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D012616:Sea Cucumbers / Q000033:anatomy & histology* / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D008670:Metals; D011480:Protease Inhibitors; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L