Crayfish Carboxypeptidase

Abstract

To help trace the evolutionary history of the pancreatic metalloexopeptidases, carboxypeptidase [EC 3.4.12.2] was isolated from the cardia of the crayfish Astacus fluviatilis. The isolation procedure included affinity chromatography on a column of potato carboxypeptidase inhibitor covalently linked to Sepharose. Approximately 25 mg of pure enzyme can be obtained by the present procedure from 50 ml of cardia fluid. The pure enzyme resembles bovine carboxypeptidase B in specificity and is inhibited both by 3-phenyllactate and by 6-aminohexanoate. The pH optimum of activity is about pH 6.5 and the isoelectric point, pH 4.0. Inhibition by typical metal chelating agents (i.e., EDTA and 1,10-phenanthroline) and neutron activation analysis indicate that, like the mammalian enzyme, crayfish carboxypeptidase is a Zn metalloenzyme. The purified enzyme migrates as a single band in cellulose acetate, disc gel and sodium dodecyl sulfate gel electrophoresis. The amino acid composition is similar to that of pancreatic carboxypeptidases except for a higher content of acidic amino acid residues. The amino acid sequence of the first 19 amino-terminal residues reveals significant homology to that of pancreatic carboxypeptidases A and B.