Purification by affinity chromatography using amastatin and properties of aminopeptidase A from pig kidney

Add time:08/14/2019    Source:sciencedirect.com

1.1. Amastatin, a specific inhibitor of aminopeptidase A (L-α-aspartyl(l-α-aspartyl(l-α-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.2.2. Aminopeptidase A which was purified 710-fold, hydrolyzed only acidic amino acid β-naphthylamide. The optimum pH and the optimum temperature was 7.5 and 45–50°C, respectively.3.3. The molecular weight was approx. 300 000 as determined by Sephadex G-200 gel filtration.4.4. 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.5.5. Amastatin inhibited aminopeptidase A in a competitive manner with l-glutamic acid β-naphthylamide, and the Ki value was calculated to be 2.5· 10−7 M. The inhibitory effect of amastatin on aminopeptidase A was not reversed by addition of Ca2+.

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