Purification and Characterization of Arylamidase from Monkey Brain1

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Abstract
Arylamidase [EC 3.4.11.2] was isolated from monkey brain extract and purified about 2100-fold in approximately 11% yield by a six-step procedure comprising extraction from monkey brain homogenate, ammonium sulfate fractionation, first hydroxylapatite chromatography, DEAEcellulose chromatography, Sephadex G-200 gel filtration and second hydroxylapatite Chromatography. The enzyme showed a single band on polyacrylamide disc electrophoresis and consisted of a single polypeptide chain, as judged by disc electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was strongly inhibited by PCMB, TPCK, and puromycin. Puromycin competitively inhibited the enzyme and the K1 value was about 5 × 10−7 M. Treatment with EDTA resulted in a loss of enzyme activity. The enzyme activity was restored by addition of Zn2+, Co2+, or Mn2+. Among various amino acid β-naphthylamides, L-alanine β-naphthylamide was most rapidly hydrolyzed and N-carbobenzoxyl-L-leucine β-naphthylamide was not hydrolyzed by this enzyme preparation. The molecular weight of the enzyme was 92,000 as determined by gel filtration on Sephadex G-200.