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.
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