Molecular cloning and characterization of Cathepsin B from a scuticociliate, Uronema marinum.
A cDNA encoding cathepsin B was cloned from the scuticociliate, Uronema marinum, which invades the olive flounder, Paralichthys olivaceus, leading to high mortalities in culturing fish. The full-length scuticociliate cathepsin B (ScCtB) gene contains an open reading frame of 1053 base pairs encoding 350 amino acids. A homology search revealed that ScCtB shares sequence identity with several piscine cathepsin Bs (48%-45%). The protein of ScCtB from U. marinum extracts was purified 12.8-fold by a one step purification process using a DEAE-Sephagel high performance liquid chromatography (HPLC) column. It had a molecular mass of 30 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, which was consistent with predicting molecular mass of mature protein (29.2 kDa) of ScCtB. The protease activity of the ScCtB enzyme was demonstrated by electrophoresis in a gelatin-acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate, Z-arginyl-arginyl-7-amido-4-methylcoumarin (Z-Arg-Arg-AMC). The optimum pH for the protease activity was 5.5. Typical of cysteine proteases, the enzyme was inhibited by trans-epoxysuccinyl-L-leucyl-amido(4-guanidino)butane (E-64) and leupeptin.
Lim
Sang Uk
SU
Faculty of Food Science and Biotechnology, Pukyong National University, Busan 608-737, Korea.
Seo
Jung Soo
JS
Kim
Moo Sang
MS
Ahn
Sang Jung
SJ
Jeong
Hyun Do
HD
Kim
Ki Hong
KH
Park
Nam Gyu
NG
Kim
Joong Kyun
JK
Chung
Joon Ki
JK
Lee
Hyung Ho
HH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016798:Ciliophora / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D005432:Flounder / Q000469:parasitology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment
D002401:Cathepsin B