N-Acetylalanine aminopeptidase, a new enzyme from human erythrocytes.
A new enzyme liberating N-acetylalanine from N-acetylalanine peptides with high specificity has been isolated from the cytosol of human erythrocytes. The N-acetylalanine aminopeptidase was purified by ammonium sulfate precipitation at 60% saturation, followed by chromatography on columns of Sephadex G-200, SP-Sephadex C-50, and DEAE-Sephadex A-50. About 2 000-fold enrichment was achieved from hemolyzed erythrocytes. The enzyme was homogeneous according to polyacrylamide disc electrophoresis and had a specific activity of 18.1 U/A280 unit. An apparent molecular weight of 300 000 +/- 15 000 was obtained from gel filtrations and was confirmed in the ultracentrifuge in "an active enzyme centrifugation" giving a corrected sedimentation value, s20w of 12 S. The pH optimum in triethanolamine/HCl buffer was around pH 8.3 with N-acetylalanine-4-nitroanilide as substrate, the Km was 0.616 mmol/l. The enzyme was stable between pH 6.0 to 8.0, but lost enzymic activity rapidly below pH 5 and with organic solvents. It is stabilized in a 0.1 M solution of ammonium sulfate. The activity was destroyed by high concentrations of chloromercuribenzoate and di(2-pyridyl)disulfide in an unspecific manner and could not be restored by cysteine. Various protein endoproteinase inhibitors are without influence on the enzymic activity. The enzyme exhibits an aminopeptidase-like activity with release of N-acetylalanine in order of decreasing activity from N-acetylalanine-4-nitroanilide, N-acetylalanyl-alanylalanine, N-acetylalanyl-tyrosyl-isoleucine, N-acetylalanylalanine, N-acetylalanyl-alanyl-alanylalanine, and N-acetylalanine ethyl ester. Several unacetylated peptides and alanine-4-nitroanilide as well as protein substrates were not hydrolyzed. The enzymic activity has not been found in the cytosolic compartment of highly purified human leucocytes. Its physiological function in erythrocytes is still unknown.
D000626:Aminopeptidases / Q000097:blood* / Q000302:isolation & purification; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000097:blood* / Q000302:isolation & purification; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013439:Sulfhydryl Reagents; D010447:Peptide Hydrolases; D000626:Aminopeptidases; C038220:acylaminoacyl-peptidase