PURIFICATION AND CHARACTERIZATION OF AN AMINOPEPTIDASE FROM TUNA (THUNNUS ALBACARES) PYLORIC CECA

An aminopeptidase was purified from a water soluble fraction of tuna p yloric caeca by heat treatment, Zn2+ fractionation, ion exchange on a DEAE cellulose column, gel filtration on Fractogel TSK-55, and immobil ized metal ion affinity chromatography (IMAC) on IDA(Cu2+/Zn2+)-Sephar ose 6B. The molecular mass of the enzyme was estimated to be 150000 on Sephacryl S-300 HR, and was found to be near 72000 by SDS-PAGE. The a minopeptidase, which is a glycoprotein rich in acidic amino acids, is optimally active at pH 8.8 and 65 degrees C. The enzyme activity was n ot affected by Mg2+, Zn2+, Ca2+, Mn2+, Co2+, PMSF, iPr(2)FP, 4-hydroxy mercuribenzoic acid, iodoacetamide, puromycin, and cysteine but it was strongly inhibited by metal chelators (EDTA and o-phenanthroline), am astatin, Hg2+, Cd2+, and CU2+, The enzyme was also inhibited by some L -amino acids. Kinetic parameters of the enzyme were determined with so me aminoacyl-p-nitroanilides and aminoacyl-beta-naphthylamides. L-Alan ine-p-nitroanilide and L-alanine-beta-naphthylamide were hydrolysed mo st rapidly while the highest hydrolytic coefficient (k(cat)/K-m) value was obtained with L-methionine-p-nitroanilide. The apoaminopeptidase was prepared and reconstitution of an active enzyme was carried out us ing metal chelating interaction chromatography on an IDA-Sepharose 6B column charged with a metal ion. Full activity was restored with Zn2Co2+, CU2+ and Al3+. Zn2+-Enzyme was the most thermostable form of the aminopeptidase. Reversal inhibition by Cu2+ and Cd2+ was also examine d. When the aminopeptidase was partially deglycosylated by a treatment with N-glycosidase F some of its physical properties differed from th at of the native enzyme: its electrophoretic mobility was reduced and its stability to denaturation by SDS and by ionic strength were lower than those of the untreated enzyme. All together, our results indicate that the tuna pyloric caeca aminopeptidase is distinct from the pepti de hydrolases characterized in the literature.