Acid proteinase activity in fish II. Purification and characterization of cathepsins B and D from Mujil auratus muscle.
Two cathepsins were detected in Mujil auratus muscle extracts. They were classified as a thiol- and aspartyl-proteinase (cathepsins B and D, respectively) on the basis of their catalytic behaviour in presence of specific inhibitors. Following extraction in 1% KCl, the proteinases were purified by autolysis, acetone fractionation, affinity chromatography, and gel permeation chromatography. The haemoglobin-agarose column chromatography allowed us to separate the two activities. Sephadex G-75 column chromatography resulted in apparent molecular weights of 25,000 (cathepsin B) and 35,000 (cathepsin D). The molecular size, together with pH-activity profiles and kinetic parameters are similar to those reported for mammalian cathepsins B and D. This was not the case with the temperature-activity profiles, the optimum temperature as well as the heat stability being higher for fish cathepsins than for those obtained from other sources. Cathepsin B was characterized by its ability to inactivate aldolase. Fluorescence quenching experiments showed that tryptophyl residues of cathepsin B were less occluded and located in a more electronegative microenvironment that those pertaining to cathepsin D.
D000818:Animals; D002401:Cathepsin B; D002402:Cathepsin D; D002403:Cathepsins / Q000302:isolation & purification* / Q000378:metabolism; D005399:Fishes / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D013816:Thermodynamics
D011480:Protease Inhibitors; D002403:Cathepsins; D002401:Cathepsin B; D002402:Cathepsin D