Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus).
The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.
Raksakulthai
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Department of Food Science and Technology, University of California at Davis, Davis, California 95616, USA.
Haard
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002268:Carboxypeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D049832:Decapodiformes; D004064:Digestive System / Q000201:enzymology*; D004151:Dipeptides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D007700:Kinetics; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D019287:Zinc Sulfate / Q000494:pharmacology
D000596:Amino Acids; D004151:Dipeptides; D012965:Sodium Chloride; D019287:Zinc Sulfate; D002268:Carboxypeptidases