Purification and properties of carp muscle cathepsin A.

To clatify the post-mortem proteolysis of fish muscle, carp muscle cathepsin A was purified and its properties were investigated. The purification was about 1, 700-fold, with a yield of 1.5%. The purification procedures were as follows: acid treatment, heat treatment, ammonium sulfate fractionation, acetone fractionation, DEAE-Sephadex A-50 chromatography, preparative electrofocusing, and Sepharose 6B gel filtration. The enzyme hydrolyzed Z-Glu-Phe more preferably than Z-Glu-Tyr, with the optimum pHwas 5.0. The Km values for Z-Glu-Phe and Z-Glu-Tyr were estimated to be 3.52mM and 4.76mM, respectively. Z-Gly-Pro was not hydrolyzed by the enzyme. On the other hand, the enzyme did not act on any protein substrates used. The enzyme activity was completely inhibited by Dip-F, PMSF, iodoacetamide, and antipain, but activated by 2-mercaptoethanol, NaI, and NaBr. Therefore, serine and cysteine residues may be involved for the activity. Pepstatin, EDTA, and o-phenanthroline had no effect on the activity. The molecular weight of the enzyme was estimated to be 36, 000 by gel filtration and the isoelectric point to be 4.6. At pH 5.0 carp muscle homogenate autolyzed considerably at 37°C for 24 hours. But cathepsin A had no electorphoretically recognizble effect on the autolysis. Therefore, the enzyme doesn ' t seem to participate directly in post-mortem degradation of fish muscle.