Purification and characterization of cathepsin L in arrowtooth flounder (Atheresthes stomias) muscle.
A predominant, heat-activated proteinase in muscle extract of arrowtooth flounder (Atheresthes stomias) was purified to 55-fold by heat treatment, followed by a series of chromatographic separations. The apparent molecular mass of the purified enzyme was 27 kDa by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteinase had high affinity and activity toward Z-Phe-Arg-NMec with K(m) and k(cat) values of 8.2 microM and 12.2/s, respectively. Activity was inhibited by sulfhydryl reagents and activated by reducing agents. The purified proteinase displayed optimal activity at pH 5.0-5.5 and 60 degrees C, respectively. Consistent with the properties of proteases from other species, the heat-activated proteinase in arrowtooth flounder can be identified as cathepsin L.
Visessanguan
Wonnop
W
BIOTEC--Central Research Unit, National Center for Genetic Engineering and Biotechnology, 113 Paholayothin Road, Klong 1, Klong Laung, Patumthani 12120, Thailand. wonnop@biotec.or.th
Benjakul
Soottawat
S
An
Haejung
H
D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D029941:Fish Proteins / Q000302:isolation & purification / Q000378:metabolism; D005399:Fishes; D005432:Flounder / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D029941:Fish Proteins; D011480:Protease Inhibitors; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L