Purification and characterization of aminopeptidase N from human plasma.
Human plasma aminopeptidase N (EC 3.4.11.2) was homogeneously purified from outdated bank plasma.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 mumol/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. SDS-polyacrylamide 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 degrees 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 Km, Vmax and Kcat values and hydrolytic coefficient (Kcat/Km) of the enzyme with L-alanyl-beta-naphthylamide as substrate were 8.7 X 10(-5) mol/l, 85.9 mumol/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.
D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D018826:CD13 Antigens; D002241:Carbohydrates / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003035:Cobalt / Q000494:pharmacology; D004355:Drug Stability; D006358:Hot Temperature; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics
D000596:Amino Acids; D002241:Carbohydrates; D003035:Cobalt; D000626:Aminopeptidases; D018826:CD13 Antigens