Crystallization and Properties of Carboxypeptidase Aγ from Porcine Pancreas

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Abstract
Carboxypeptidase Aγ from porcine pancreas was purified to homogeneity by ammonium sulfate fractionation, autolysis, batch absorption and elution from DEAE‐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 38900. The amino‐terminal sequence of the porcine carboxypeptidase Aγ was Asn‐Tyr‐Ala‐Thr‐Tyr‐His‐Thr‐Leu‐GluGlu‐Ile‐Tyr‐Asp‐Phe‐Met‐Asp‐Ile‐Leu‐Val‐Ala‐Glu‐His‐Pro‐Gln‐Leu‐ which was highly homologous to that of bovine carboxypeptidase AYγ. The purified enzyme was characterized with respect to isoelectric point (4.3). Km for Nα‐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 iEDTA and o‐phenanthroline.Among substrates such as Cbz‐Gly‐lPhe, Nα‐benzoylglycyl‐l‐arginine, various kinds of amino acid esters, casein and elastin, porcine carboxypeptidase Aγ 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.

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