PURIFICATION AND CHARACTERIZATION OF PACIFIC WHITING PROTEASES

Two forms (P-I and P-II) of a protease were purified to 560- and 21.5-fold from the sarcoplasmic fluid of Pacific whiting (Merluccius productus). The enzymes were assayed by hydrolysis of azocasein at 55 degrees C and pH 5.5. Both forms were composed of a single polypeptide of M(r) 28 800; however; P-I also contained two low M(r) components which were removed by low pH treatment prior to size exclusion chromatography. P-I and P-II showed pH optima at 5.5 and 6.0 and temperature optima of 55 and 60 degrees C, respectively. The enzymes had an identical isoelectric point of 4.91 with a minor band at 4.94. The proteases had high activity toward Z-Phe-Arg-NMec- and casein. The activity of both forms was inhibited by all sulfhydryl reagents tested as well as the cathepsin L-specific inhibitor Z-Phe-Phe CHN2 and activated by reducing agents, suggesting the enzymes are forms of cathepsin L.