Crystallization and properties of carboxypeptidase A gamma from porcine pancreas.
Carboxypeptidase A gamma from porcine pancreas was purified to homogeneity by ammonium sulfate fractionation, autolysis, batch absorption and elution from DEAF-Sephadex, and crystallization. The overall purification was about 32-fold with a yield of 31% and the specific activity of the purified protein was 108 units/mg protein. The apparent relative molecular mass determined by gel filtration on a Sephadex G-200 column was 38 900. The amino-terminal sequence of the porcine carboxypeptidase A gamma was Asn-Tyr-Ala-Thr-Tyr-His-Thr-Leu-Glu-Glu-Ile-Tyr-Asp-Phe-Met-Asp-Ile-Leu-Val-Ala -Glu-His-Pro-Gln-Leu- which was highly homologous to that of bovine carboxypeptidase A gamma. The purified enzyme was characterized with respect to isoelectric point (4.3). Km for N alpha-carbobenzoxyglycyl-L-phenylalanine (Cbz-Gly-LPhe) (20 mM), amino acid composition, pH optimum, pH stability, stability at different temperatures and effect of drying. The enzyme contained 1.01 mol zinc/mol and was inhibited by chelating agents such as EDTA and o-phenanthroline. Among substrates such as Cbz-Gly-LPhe, N alpha-benzoylglycyl-L-arginine, various kinds of amino acid esters, casein and elastin, porcine carboxypeptidase A gamma showed an enzymatic activity only towards Cbz-Gly-LPhe and casein. These data are in good agreement with the substrate specificity of bovine carboxypeptidase A.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002268:Carboxypeptidases / Q000302:isolation & purification*; D043422:Carboxypeptidases A; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine; D013696:Temperature; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D015032:Zinc