Partial Resolution of Brain Arylamidases and Aminopeptidases

Abstract

Abstract A procedure is described for the partial resolution of enzymes in the rat brain that hydrolyze aminoacylated naphthylamines and aminopeptidase substrates. These enzymes were partially purified by (NH4)2SO4 precipitation, gel filtration, and diethylaminoethyl cellulose resolution. Three distinct peaks were resolved on DEAE-cellulose. The first peak showed marked specificity for the tripeptide Leu-Gly-Gly; the second was specific for arginyl- and lysyl-β-naphthylamides; the third was active with both basic and neutral amino acid naphthylamides. Study of the arylamidases showed that their properties are distinct from those of the exo- and endopeptidases. Purified arylamidases were shown to be dependent on metal ions for activity. Treatment with ethylenediaminetetraacetate resulted in loss of activity, which could be restored by the addition of Zn2+, Co2+, Mn2+, Ca2+, Mg2+, or La3+. The values of Km and the optimal pH varied with the different arylamide analogues. For leucyl-β-naphthyl-amide the value of Km was 8 x 10-4 m at the optimal pH of 6.5; for arginyl- and lysyl-β-naphthylamides, the values were 5 x 10-4 m at pH 6.5 and 1.3 x 10-4 m at pH 8.0, respectively. Arylamidases required dithiothreitol for stability; activity was increased by cysteine and β-mercaptoethanol, and inhibited by p-chloromercuribenzoate. The properties of the brain arylamidases were distinct from those of brain aminotripeptidase and hog kidney leucine aminopeptidase (EC 3.4.1.1). Unlike the arylamidases, the other enzymes were not affected by puromycin.