PURIFICATION AND CHARACTERIZATION OF A NOVEL CYSTEINE PROTEINASE FROM MACKEREL (SCOMBER AUSTRALASICUS)

A novel cysteine proteinase from mackerel muscle was purified to electrophoretic homogeneity using HiLoad DEAE-Sepharose, HiLoad S Sepharose, and FPLC Superdex 75 chromatographies. The molecular weight of purified proteinase was 99 000 estimated by Superose 12 gel filtration. This proteinase appeared as two protein bands of 35 000 (designated ''a'') and 23 000 (designated ''b'') on SDS-PAGE. This purified proteinase, accordingly, identified to be a trimer of the form a(2)b. It hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA but not Z-Arg-MCA and L-Arg-MCA. The optimal pH and temperature for the hydrolysis of Z-Arg-Arg-MCA were 6.0 and 35 degrees C, respectively. The proteinase was stable at pH 5.5-6.0 but was unstable when the pH was higher than 7.0. The inactivation rate constant (K-D) at 50 degrees C was 3.8 x 10(-1) min(-1). This proteinase was activated by dithiothreitol, cysteine, glutathione, and beta-mercaptoethanol. The thiol-dependent proteolytic activity was substantially inhibited by 1-(L-trans-epoxysuccinylleucylamino)-4-guanidinobutane, antipain, iodoacetic acid, leupeptin, tosyllysine chloromethyl ketone, Zn2+, Cd2+, Co2+, Ni2+, Cu2+, Hg2+, Fe2+, and Fe3+. These results suggest that the cysteine residue is required for the catalytic activity of this proteinase.