Purification and partial characterization of a carboxypeptidase from the limpet (Patella vulgata)

A carboxypeptidase A-like enzyme has been purified to apparent homogeneity from commercially available acetone powder from the visceral hump of the limpet, Patella vulgata. A two-step procedure involving affinity chromatography on ?-amino-N-caproyl-d-phenylalanine-Sepharose and gel filtration resulted in a 3000-fold purification with an 80% yield. The enzyme is a single polypeptide chain of M_r = 40,000 and exhibits both peptidase and esterase activities, which are characterized by dramatic excess substrate inhibition. Inhibition studies suggest that a metal ion is required for activity and demonstrate that the affinity label, N-bromoacetyl-N-methyl-l-phenylalanine, and a polypeptide carboxypeptidase inhibitor from potatoes (apparent K_i approx. 2 nm) are effective against the limpet enzyme.