Aminopeptidase PC from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica

Homogeneous aminopeptidase PC was isolated with yield 67% and purification degree 237 from the hepatopancreas of the Kamchatka crab Paralithodes camts hatica by ion-exchange chromatography on DEAE-Sepharose, hydrophobic chroma tography on Phenyl-Sepharose, and gel-filtration on Sephadex G-150. The enz yme is a homodimer with a molecular mass 220 kD (110 x 2). Aminopeptidase P C has pI = 4.1. It hydrolyzes Leu-pNA optimally at pH 6.0 and at the optimu m temperature 36-40 degrees C; in the presence of Ca2+ the enzyme is stable at pH 5.5-8.0. Aminopeptidase PC is activated by Ca2+, Mg2+, and Fe2+; it is completely inhibited by EDTA, o-phenanthroline, and bestatin. The enzyme contains four Zn atoms per molecule and is therefore a metalloaminopeptida se. The aminopeptidase PC can effectively cleave N-terminal Arg and Lys res idues as well as Leu, Phe, and Met residues. K-m and k(cat), values for hyd rolysis of Leu-pNA were 0.075 mM and 0.19 sec(-1) and for hydrolysis of Arg -pNA 0.078 mM and 0.48 sec(-1), respectively. D-Amino acid residues cannot be cleaved. Thus, aminopeptidase PC of the Kamchatka crab has a mixed subst rate specificity which is characteristic of some microbe aminopeptidases. i ts N-terminal sequence ESVEIELPEGLSPLV is 46% coincident with that of yeast vacuolar aminopeptidase YSCA.