Isolation and characterization of aminotripeptidase from monkey brain.

Aminotripeptidase [EC 3.4.11.4] was purified from monkey brain by a five-step procedure comprising extraction from brain homogenate, ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxylapatide chromatography, and Sephadex G-200 gel filtration. A purification of 1,100-fold over the homogenate was achieved and the yield was 12%. The purified enzyme appeared to be homogeneous on polyacrylamide gel electrophoresis at pH 8.9. The amino acid composition of the enzyme resembled that of the pig kidney enzyme. The molecular weight of the enzyme was estimated to be about 65,000 by gel filtration on Sephadex G-200 and 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pH optimum for L-leucyl-glycyl-glycine was about pH 7.5. The enzyme hydrolyzed only tripeptides to yield the NH2-terminal residues as free amino acids and the residual dipeptides. The enzyme did not show activities of arylamidase or carboxypeptidases A and B. The enzyme was inhibited by PCMB, o-phenanthroline, and bestatin. The inhibition by bestatin was competitive and the K1 value was calculated to be 5 X 10(-7) M.