1Department of Brewing and Fermentation, Junior College of Tokyo University of Agriculture, Sakuragaoka, Setagaya, Tokyo 156-8502, 2Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, Sakuragaoka, Setagaya, Tokyo 156-8502, Japan
Aminopeptidases of the intestine of Tilapia nilotica were purified by ammonium sulfate precipitation, followed by trypsin inhibitor-Sepharose4B affinity chromatography, DEAE-cellulose ion-exchange chromatography and polyexchanger PBE 94 chromatofocusing.
Two different Aminopeptidases were obtained in the pure state and tentatively designated aminopeptidase I and II. Each of the two aminopeptidases was found to be a single band when examined by native polyacrylamide gel electrophoresis. The purifications of aminopeptidase I and II were 63- and 79-fold from the crude extract, respectively.
Aminopeptidase I and II had molecular weight of 240,000 and 300,000, respectively. Aminopeptidase I showed the highest activity at pH7.0 and 45°C. Aminopeptidase II showed the highest activity at pH6.5 and 50°C. Aminopeptidase I specifically was found to be able to hydrolyze Ala-pNA, while II was found to be able to hydrolyze Leu-pNA.
Two enzymes were strongly inhibited by DFP, TPCK, EDTA and o-phenanthroline. The activities of two enzymes inactivated by EDTA were regenerated by the addition of Zn2+, Co2+, Mg2+ and Cu2+.