Purification and Characterization of Aminopeptidase N from Human Plasma

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Abstract
Human plasma aminopeptidase N (EC 3.4.11.2) was homogeneously purified from outdated bank plasma. Purification procedures included ammonium sulfate fractionation, immunoaffinity chromatography, DEAE-cellulose column chromatography, hydroxyapatite column chromatography and Sephadex G-200 gel filtration. The final recovery of the enzyme was 18% and its specific activity was 71.6 μmol/min/mg protein. SDS-polyacrylamide gel disc electrophoresis and analytical ultracentrifugation showed the homogeneity of the enzyme. Equilibrium ultracentrifugation showed a molecular weight of 210,800. SDSpolyacrylamide gel disc electrophoresis indicated that the enzyme was a dimer consisting of two identical subunits. The isoelectric point of the enzyme was 3.9 at 4 °C. The amino acid composition of the enzyme was very similar to those of aminopeptidase N from human kidney, small intestine, and placenta which we have reported previously. Neutral sugar accounted for 11.6%. The K(m), V(m)ax and K(cat), values and hydrolytic coefficient (K(cat)/K(m)) of the enzyme with L-alanyl-ß-naphthylamide as substrate were 8.7 X 10-^5 mol/1, 85.9 μmol/min/ mg protein, 303/s and 3,483/mmol/l/s, respectively. The enzyme was activated by cobalt ions and markedly inhibited by amastatin. Plasma aminopeptidase N was immunologically indistinguishable from kidney aminopeptidase N.