Isolation and properties of carboxypeptidase from the Kamchatka crab Paralithodes camtshatica
Rudenskaia, G.N.; Kupenko, O.G.; Isaev, V.A.; Stepanov, V.M.; Dunaevskiĭ, I.E.
Bioorganicheskaia Khimiia 21(4): 249-255
1995
ISSN/ISBN: 0132-3423 PMID: 7786315 Document Number: 447853
Homogeneous carboxypeptidase PC from a hematopancreas of kamchatka crab Paralithodes camtshatica was obtained by means of an affinity chromatography on sorbents containing arginine, protamine hydrolysate, and phenylalanine as ligands with an yield 23% and purification degree 37.4. The isolated enzyme has a molecular mass 34 kDa, as evidenced by an SDS-PAGE; pI 3.1; an optimum pH 6.5, as estimated for hydrolysis of Dnp-Ala-Ala-Arg; pH-stability range 5-8 in the presence of Ca2+; a temperature optimum 55 degrees C; and Km 0.4 mM. The carboxypeptidase is activated by Co2+ and Ca2+ ions and is inhibited by EDTA and o-phenanthroline, and therefore, it is a metallocarboxypeptidase. The enzyme can effectively split off C-terminal residues Phe and Tyr, as well as Arg and Lys. Residues Pro, Glu, and Asp cannot be split off, and they stop the cleaving of a preceding bond. Thus, the carboxypeptidase PC of kamchatka crab has a mixed substrate specificity, which is characteristic of carboxypeptidase from crawfish and of microbial carboxypeptidases T and SG. The new carboxypeptidase has an amino acid composition Asp41Thr24Ser22Glu32Pro15Gly32Ala291/2Cys5Val19Met8Ile14Leu20Ty r18Phe8Lys7His4 Arg8Trp4. The N-terminal sequence of the enzyme demonstrate a 40% homology with the N-terminal sequence of carboxypeptidase from crawfish.