Characterization of proteins from Ascaris lumbricoides which bind specifically to carboxypeptidase

Inhibitors of the peptidase and esterase activities of carboxypeptidases A and B have been isolated from extracts of Ascaris lumbricoides var suis. These proteins were obtained by treatment of the aqueous extracts at low pH, precipitation with ammonium sulfate, molecular sieving on Bio-Gel P-4, and chromatography on DEAE-cellulose. The inhibitors were resolved into three homogeneous peaks on CM-cellulose. These components, CM-A, CM-B, and CM-C, have constant specific activity and were recovered in a 41% yield. They moved as single bands when subjected to electrophoresis at high or low pH on polyacrylamide gels and they have similar amino acid compositions. Methionine, tyrosine, and cysteine are absent from each of the inhibitors. The 65 residues of CM-B suggest a minimum molecular weight of 7530, in close agreement to the value of 7600 +/- 200 determined on a Bio-Gel P-100 column. Each of the proteins has the same NH2-terminal residues, NH2-Asx-Glx-Val-Glx- and the same COOH-terminal residue, leucine. A plot of per cent acrylamide versus log relative mobility suggests that the three proteins are charge isomers. CM-B appears to be stable to high NaCl concentrations, extremes of pH, high temperatures, and digestion by intestinal proteases. Carboxypeptidase C, carboxypeptidase N, and yeast protease C are not inhibited by CM-B. However, the exopeptidase and esterase activities of human carboxypeptidase A are inhibited. The inhibitors appear to bind to bovine carboxypeptidase A with an atypical stoichiometry. Two moles of CM-B inhibitor bind to 1 mol of enzyme. The evidence is: (a) a demonstrated purity of bovine carboxypeptidase A, (b) minimal and maximal inhibitor molecular weights by different methods, of 7600 and 8300, and (c) a maximum specific activity of apparently homogeneous inhibitors which is 50% of that predicted for unit stoichiometry.