Purification and crystallization of human carboxypeptidase A.
Human carboxypeptidase A has been isolated from activated pancreatic juice by means of affinity chromatography employing the competitive inhibitor benzylsuccinic acid as an affinity ligand. The structural and functional features of the human and bovine enzymes are quite analogous. The molecular weights of human and bovine carboxypeptidases A are virtually identical, their amino acid compositions are similar, both contain 1 g-atom of zinc/mole, and the activities of both are restored by addition of zinc to the apoenzyme. The inhibition of human carboxypeptidase by chelating agent is reversed by either dilution or addition of a metal such as Cu2+. When other metals are substituted for the native zinc, peptidase activity of the human metallocarboxypeptidases follows the order: cobalt greater than nickel greater than manganese greater than cadmium, while the sequence for esterase activities is: manganese greater than cobalt = cadmium greater than nickel. The latter sequence differs from that observed for the bovine enzyme. Human carboxypeptidase A crystallizes after dialysis at low ionic strength. Hydrolysis of the dipeptide carbobenzoxyglycyl-L-phenylalanine and of the ester benzoylglycyl-L-alpha-hydroxy-beta-phenyllactate exhibits kinetic anomalies, but that of their longer homologues does not. Chemical modifications with tyrosine reagents alters esterase and peptidase activities. The affinity chromatographic method here described should greatly facilitate future studies of this enzyme from human and other sources.
D000596:Amino Acids / Q000032:analysis; D002268:Carboxypeptidases / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D003460:Crystallization; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008855:Microscopy, Electron, Scanning; D010179:Pancreas / Q000201:enzymology*; D011487:Protein Conformation
D000596:Amino Acids; D007527:Isoenzymes; D002268:Carboxypeptidases