ISOLATION AND CHARACTERIZATION OF A CARBO XYPEPTIDASE FROM A KAMCHATKA CRAB PARALITHODES CAMTSHATICA

Homogeneous carboxypeptidase PC from a hematopancreas of kamchatka cra b Paralithodes camtshatica was obtained by means of an affinity chroma tography on sorbents containing arginine, protamine hydrolysate, and P henylalanine 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 Dn p-Ala-Ala-Arg; pH-stability range 5 - 8 in the presence of Ca2+; a tem perature optimum 55 degrees C; and K-m 0.4 mM. The carboxypeptidase is activated by Co2+, and Ca2+ ions and is inhibited by EDTA and o-phena nthroline, 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 splitted off, and th ey stop the cleaving of a preceding bond, Thus, the carboxypeptidase P C of kamchatka crab has a mixed substrate specificity, which is charac teristic of carboxypeptidase from crawfish and of microbial carboxypep tidases T and SG. The new carboxypeptidase has an amino acid compositi on e(14)Leu(20)Try(18)Phe(8)Lys(7)His(4)Arg(8)Trp(4). The N-terminal s equence of the enzyme demonstrate a 40% homology with the N-terminal s equence of carboxypeptidase from crawfish.