Open AccessCrystallization and Properties of Carboxypeptidase A γ from Porcine Pancreas
Author(s) -
KOIDE Atsushi,
YOSHIZAWA Masayuki,
KURACHI Kotoku
Publication year - 1981
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1981.tb06349.x
Subject(s) - chemistry , carboxypeptidase a , carboxypeptidase , sephadex , isoelectric point , chromatography , biochemistry , casein , size exclusion chromatography , amino acid , phenylalanine , enzyme
Carboxypeptidase A γ 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 A γ 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 AY γ . The purified enzyme was characterized with respect to isoelectric point (4.3). K m for N α ‐carbobenzoxyglycyl‐ l ‐phenylalanine (Cbz‐Gly‐ l Phe) (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‐ l Phe, N α ‐benzoylglycyl‐ l ‐arginine, various kinds of amino acid esters, casein and elastin, porcine carboxypeptidase A γ showed an enzymatic activity only towards Cbz‐Gly‐ l Phe and casein. These data are in good agreement with the substrate specificity of bovine carboxypeptidase A.