Purification and characterization of carboxypeptidase A from rat skeletal muscle

Carboxypeptidase A (EC 3.4.17.1) has been purified 44 000-fold in 33% yield from rat skeletal muscle by a four-step procedure. Purification in the presence of dichlorovinyl dimethyl phosphate conveniently inactivates an accompanying chymotrypsin-like enzyme and other serine protease(s) to ensure isolation of pure carboxypeptidase A free of polypeptide contaminants. The enzyme preparation consists of two components with molecular weights of approximately 39 300 and 37 800. The rat muscle carboxypeptidase is very similar to bovine pancreatic carboxypeptidase A in terms of (1) substrate specificity, (2) kinetics and molecular activity, (3) influence of metal ions on catalysis, (4) interaction with inhibitors, (5) effects of ionic strength on activity, and (6) stability and activity as functions of pH. Both muscle and pancreatic carboxypeptidases exhibit enhanced esterolytic activity when assayed in the presence of a variety of indoles and imidazoles or after incubation at relatively high concentrations of MnSO4. The muscle enzyme is substantially less stable than its pancreatic homologue, and in impure preparations is very much less soluble. The latter property is attributable to a binding substance present in such preparations which renders muscle but not pancreatic carboxypeptidase A insoluble until ionic strength is increased to values near 2 M.