title: Molecular cloning and characterization of Cathepsin B from a scuticociliate, Uronema marinum.
abstract: 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.
authors.affiliation: Faculty of Food Science and Biotechnology, Pukyong National University, Busan 608-737, Korea.
authors.forename: Sang Uk
authors.initials: SU
authors.lastname: Lim
authors.forename: Jung Soo
authors.initials: JS
authors.lastname: Seo
authors.forename: Moo Sang
authors.initials: MS
authors.lastname: Kim
authors.forename: Sang Jung
authors.initials: SJ
authors.lastname: Ahn
authors.forename: Hyun Do
authors.initials: HD
authors.lastname: Jeong
authors.forename: Ki Hong
authors.initials: KH
authors.lastname: Kim
authors.forename: Nam Gyu
authors.initials: NG
authors.lastname: Park
authors.forename: Joong Kyun
authors.initials: JK
authors.lastname: Kim
authors.forename: Joon Ki
authors.initials: JK
authors.lastname: Chung
authors.forename: Hyung Ho
authors.initials: HH
authors.lastname: Lee
mesh_terms: 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
chemical_list: D002401:Cathepsin B