M. Hajjou et Y. Legal, PURIFICATION AND CHARACTERIZATION OF AN AMINOPEPTIDASE FROM TUNA (THUNNUS ALBACARES) PYLORIC CECA, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1204(1), 1994, pp. 1-13
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.