N-Acetylaminoacyl-p-nitranilidase from human placenta. Purification and some properties.

An enzyme hydrolyzing N-acetylaminoacyl-p-nitroanilides has been isolated from mature human placentae by a six-step procedure comprising extraction from a placenta homogenate, ammonium sulfate fractionation, treatment with isopentyl alcohol, chromatography on CM-Sephadex, protamine sulfate precipitation and gel filtration on an Ultrogel AcA 34 column. About 2500-fold enrichement was achieved from placenta homogenate. The purified enzyme preparation showed a single band on polyacrylamide disc electrophoresis. The molecular weight was estimated to be 380,000 by gel filtration. Placental extracts contain two main isoenzymes of pI 3.9 and 4.5 respectively. Activity was strongly inhibited by chloromercuribenzoate, slightly inhibited by Ca2+ and cysteine; no activation could be detected. The enzyme exhibits an exopeptidase-like activity towards acetyl-dipeptides with a certain specifity towards N-acetylalanyl-alanine; N-acetylalanine-p-nitroanilide, however, is hydrolyzed four times faster. With N-acetylalanine-p-nitroanilide as substrate the pH optimum was 8.0--8.2; Km was 2.13 mmol/l. N-Acetylleucine-p-nitroanilide and N-acetyltyrosine-p-nitroanilide were split slowly; N-acetylalanyl-alanyl-alanine-p-nitroanilide, N-butyloxycarbonyl-alanyl-alanine-p-nitroanilide, unsubstituted analogous aminoacyl-p-nitroanilides and several protein substrates were not hydrolyzed. The functions of the enzyme are still unknown.