L-Isoleucine 4-nitroanilide

Six aminopeptidases differing in enzymic specificity against various L-amino acid-4-nitroanilides were detected and isolated from the cytosol of leucocytes collected from the buffy coat of human blood. The different enzymes were separated by one step of chromatography on DEAE-Sephacel and were further purified by gel filtration on Sephacryl S-300. The main aminopeptidases of the cytosol were designated aminopeptidases 1, 2, 4 and 5 (AP 1, AP 2, AP 4, AP 5) on the basis of their elution sequence from the first ion-exchange chromatography column on DEAE-Sephacel. Aminopeptidase 1 appeared to be a strongly sulfhydryl-dependent leucine aminopeptidase, activatable by thiol reagents. The enzyme was inhibited by p-chloromercuribenzoate. Its molecular mass was estimated to be 150 kDa. Aminopeptidase 2 showed high specificity for proline-4-nitroanilide. This enzyme was inhibited by p-chloro-mercuribenzoate and bestatin. It exhibited a molecular mass of 70 kDa. Aminopeptidase 4 designated the activities of two different enzymes of apparent molecular masses of 220 and 70 kDa which could be further separated by gel filtration. Aminopeptidase 5 exhibited the properties alike aminopeptidase B with high specific hydrolytic activity against the 4-nitroanilides of lysine and arginine. The molecular mass was estimated to be 90 kDa. Aminopeptidase 3 was a minor component in the cytosol and could be identified as an extracellular leucocyte plasma membrane constituent. The enzyme exhibited properties of a metallo proteinase and could be inhibited by EDTA. The molecular mass was estimated to be 250 kDa.

An aminopeptidase was purified about 1,700-fold from seeds of Japanese apricot (Prunus mume Sieb.) by a seven-step procedure comprising extraction from seeds, ammonium sulfate fractionation, DEAE-cellulose chromatography, first and second DEAE-Sepharose chromatography, hydroxyapatite chromatography, and Sephadex G-200 gel filtration. The purified enzyme was shown to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56,000 by gel filtration on Sephadex G-200 and the isoelectric point was 4.9. The pH optimum for L-leucine beta-naphthylamide was between pH 6.5 and 7.0, and the enzyme was stable in the pH 5.0 to 8.3 region and up to 50 degrees C. The enzyme hydrolyzed a variety of aminopeptidase substrates with a free alpha-amino group. Of the amino acid beta-naphthylamides, the enzyme was highly specific for substrates with a hydrophobic side chain in the amino terminal residue. The enzyme was strongly inhibited by p-chloromercuribenzoate, heavy metals, diethyl pyrocarbonate, and photooxidation with methylene blue, but was not affected by thiol compounds, peptidase inhibitors of microbial origin, such as bestatin and puromycin, or metal chelating agents. No activation of the enzyme by metal ions was observed. These results suggest that the enzyme is a true aminopeptidase in which cysteine and histidine residues participate in the catalytic process, and is not classifiable as a metalloenzyme.

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.