Abstract
Two proteolytic enzymes designated as J-I and J-II were obtained from the extracts of jellied Pacific hake Merluccius productus muscle. Both enzymes were purified through a sequence of ammonium sulfate fractionation, chromatography on DEAE-cellulose, gel filtration on Sephadex G-100 and Bio gel P-30, and isoelectric focusing method. The final yields of the proteolytic activities for the purified J-I and J-II were 4.8 and 3.6%, with 138- and 174-fold purification, respectively. The molecular weights estimated by gel filtration were 14, 500 for J-I and 26, 000 for J-II. Their pH optima were at pH 3.0 with hemoglobin and pH 6.0 with casein. Both enzymes were inhibited by ICH2COOH, PCMB, E-64, antipain, and leupeptin, but not EDTA and pepstatin. They easily hydrolyzed Pyr-Phe-Leu-pNA and Z-Phe-Arg-MCA, but not Z-Ala-Ala-Leu-pNA, Suc-Ala-Ala-Ala-pNA, or Arg-pNA/MCA. Treatment of aldolase with each enzyme inactivated up to about 70% of the initial aldolase activity with fructose-1, 6-bisphosphate as a substrate.
Two enzymes jellied the normal muscle of Pacific hake at 40°C for 10h. Under this condition, the degradation of the muscle tissue was observed by light microscopy.
These results indicate that the proteases (J-I and J-II) are responsible for jellification of the muscle of Pacific hake and are classified as cathepsin L-like cysteine protease.
