Abstract
An arylamidase (aminopeptidase N) was purified from Escherichia coli HB101 about 300-fold by sequential chromatographies on DEAE-Sephadex, hydroxyapatite and Sephadex G-150, assaying the arylamidase and peptidase activities using L-leucyl-β-naphthylamide and Met-Ala-Ser as substrates, respectively. Both enzyme activities were inseparable throughout the purification. The purified enzyme appeared homogeneous by disc gel electrophoresis, and the two activities were detected at the same protein band. The enzyme was most active at pH 7.5 and had a molecular weight of 80, 000 and a pi 5.7. The enzyme was inhibited by o-phenanthroline, p-chloromercuribenzoate, puromycin, bestatin, and amastatin. Both the activities of arylamidase and peptidase were inhibited in parallel by o-phenanthroline and PCMB. These lost activities were restored by the addition of metal ion and 2-mercaptoethanol, respectively. By these experimental results, aminopeptidase N was concluded to possess not only arylamidase activity but also peptidase activity like aminopeptidase M from mammalian tissues, contrary to the results reported previously by several workers.
