Purification by affinity chromatography using amastatin and properties of aminopeptidase A from pig kidney.
1. Amastatin, a specific inhibitor of aminopeptidase A (L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7), was linked to an agarose matrix and by this affinity chromatography aminopeptidase A of pig kidneys was purified as a single protein shown by acrylamide gel electrophoresis. 2. Aminopeptidase A which was purified 710-fold, hydrolyzed only acidic amino acid beta-naphthylamide. The optimum pH and the optimum temperature was 7.5 and 45-50 degrees C, respectively. 3. The molecular weight was approx. 300 000 as determined by Sephadex G-200 gel filtration. 4. The activity of aminopeptidase A was not affected by sulfhydryl agents, S-S dissociating agents and serine proteinase inhibitor, but was inhibited strongly by metal chelating agents, and enhanced by alkaline earth metals. 5. Amastatin inhibited aminopeptidase A in a competitive manner with L-glutamic acid beta-naphthylamide, and the Ki value was calculated to be 2.5 x 10(-7) M. The inhibitory effect of amastatin on aminopeptidase A was not reversed by addition of Ca2+.
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D000900:Anti-Bacterial Agents; D002846:Chromatography, Affinity; D043384:Glutamyl Aminopeptidase; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D009842:Oligopeptides; D010455:Peptides; D013552:Swine
D000900:Anti-Bacterial Agents; D009842:Oligopeptides; D010455:Peptides; C016977:amastatin; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase