Tokioka-Terao M, Hiwada K, Kokubu T
Enzyme. 1984;32(2):65-75. doi: 10.1159/000469453.
Human plasma aminopeptidase N (EC 3.4.11.2) was homogeneously purified from outdated bank plasma.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.
人血浆氨肽酶N（EC 3.4.11.2）从过期库存血浆中得到了均一纯化。纯化步骤包括硫酸铵分级分离、免疫亲和层析、DEAE-纤维素柱层析、羟基磷灰石柱层析和Sephadex G-200凝胶过滤。该酶的最终回收率为18%，其比活性为71.6 μmol/min/mg蛋白。SDS-聚丙烯酰胺凝胶圆盘电泳和分析超速离心显示该酶具有均一性。平衡超速离心显示其分子量为210,800。SDS-聚丙烯酰胺凝胶圆盘电泳表明该酶是由两个相同亚基组成的二聚体。该酶在4℃时的等电点为3.9。该酶的氨基酸组成与我们之前报道的人肾、小肠和胎盘来源的氨肽酶N非常相似。中性糖占11.6%。以L-丙氨酰-β-萘酰胺为底物时，该酶的Km、Vmax、Kcat值和水解系数（Kcat/Km）分别为8.7×10⁻⁵ mol/l、85.9 μmol/min/mg蛋白、303/s和3,483/mmol/l/s。该酶被钴离子激活，并被氨抑素显著抑制。血浆氨肽酶N与肾氨肽酶N在免疫学上无法区分。
