PURIFICATION AND CHARACTERIZATION OF A PROTEINASE IDENTIFIED AS CATHEPSIN-D FROM TILAPIA MUSCLE (TILAPIA-NILOTICA X TILAPIA-AUREA)

For understanding the characteristics of lysosomal enzymes, cathepsin D was first purified by heat treatment, concanavalin A-Sepharose and Sephadex G-150 chromatographies, and finally preparative electrophoresis. A purification of 244-fold against the crude extract was achieved. The recovery was 2%. The purified enzyme appeared to be electrophoretically homogeneous and had a molecular weight of 55 000. Inhibitor study and molecular weight determination indicated this enzyme to be an aspartic proteinase, cathepsin D. The optimal pH and temperature were 3.5 and 37-degrees-C, respectively. This proteinase was inhibited by Hg2+ and Fe3+ but activated by Ca2+, Ni2+, and Mg2+ and slightly activated by Zn2+ and Cd2+. The purified cathepsin D was completely inhibited by pepstatin, partially inhibited by p-(chloromercuri)benzoate (PCMB), and ethylenediaminetetraacetic acid (EDTA), and almost not affected by 2-mercaptoethanol.