1. 1. Four distinct carboxypeptidases (EC 3.4.12.-) were shown to exist in rat liver lysosomes: cathepsin A, catheptic carboxypeptidase B, catheptic carboxypeptidase C, and catheptic carboxypeptidase G. These carboxypeptidases were distinguished on the basis of substrate specificity, thiol requirement, and separation by Sephadex G-100 column chromatography. The Km and pH optimum for each carboxypeptidase were determined. 2. 2. From the Sephadex G-100 elution profile, two peaks of cathepsin A activity were found. The major cathepsin A peak, cathepsin A1, was further purified by ion-exchange chromatography on DEAE-cellulose. 3. 3. The highly purified cathepsin A1 fraction hydrolyzed N-benzyloxycarbonyl (Cbz)-Glu-Phe, Cbz-Gly-Phe, and Ac-Phe-Tyr, which suggests that these hydrolytic activities were due to the same enzyme. 4. 4. Catheptic carboxypeptidase B and catheptic carboxypeptidase G eluted from the Sephadex G-100 column along with cathepsin B2. This suggests that cathepsin B2 may have carboxypeptidase activity. 5. 5. The purification of the lysosomal carboxypeptidases over homogenate activities was: cathepsin A1, 1280-fold; catheptic carboxypeptidase B, 220-fold; and catheptic carboxypeptidase G, 920-fold. Catheptic carboxypeptidase C was purified 16-fold over light-mitochondrial supernatant activity.
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