人胎盘和肾脏中膜结合芳基酰胺酶的分离与特性研究

Isolation and characterization of membrane-bound arylamidases from human placenta and kidney.

作者信息

Hiwada K, Ito T, Yokoyama M, Kokubu T

出版信息

Eur J Biochem. 1980 Feb;104(1):155-65. doi: 10.1111/j.1432-1033.1980.tb04411.x.

DOI:10.1111/j.1432-1033.1980.tb04411.x

Abstract

Membrane-bound arylamidases (EC 3.4.11.2) from human placenta and kidney were purified. The enzymes were solubilized from membrane fractions using trypsin. Purification procedures included DEAE-cellulose column chromatography, concanavalin-A--Sepharose 4B column chromatography, hydroxyapatite column chromatography and Sephadex G-200 gel filtration. The final recoveries of placental and kidney enzymes were 22.0% and 20.8% respectively. Polyacrylamide gel electrophoresis, analytical isoelectric focusing, immunoelectrophoresis and ultracentrifugation indicated the homogeneity of both purified enzymes. Equilibrium ultracentrifugation showed molecular weights of 193000 for placental enzyme and 211000 for kidney enzyme. Electrophoresis of the enzymes under denaturing conditions on dodecylsulfate/polyacrylamide gel indicated that each enzyme was a dimer consisting of two identical subunits. Isoelectric points of placental and kidney enzymes were 4.20 and 4.32 at 4 degrees C, respectively. The amino acid compositions of the two membrane-bound arylamidases were similar. The carbohydrate accounted for 18.2% of placental enzyme and 17.4% of kidney enzyme. The purified placental enzyme had a specific activity of 60.1 mumol min-1 (mg protein)-1 and the kidney enzyme had a specific activity of 96.4 mumol min-1 (mg protein)-1. Kcat values of placental and kidney enzymes were 187.8 s-1 and 328.6 s-1, respectively. The hydrolytic coefficient (Kcat/Km) of placental enzyme for L-alanyl-beta-naphthylamide was 2159 mM-1 s-1 and that of kidney enzyme was 3778 mM-1 s-1. Rabbit antibodies against placental and kidney membrane-bound arylamidases inhibited the activities of the corresponding antigens, but the inhibitions were never complete. Three membrane-bound arylamidases from placenta, kidney and renal cell carcinoma were immunochemically indistinguishable.

摘要

从人胎盘和肾脏中纯化出膜结合芳基酰胺酶（EC 3.4.11.2）。使用胰蛋白酶从膜组分中溶解这些酶。纯化步骤包括DEAE - 纤维素柱色谱、伴刀豆球蛋白A - 琼脂糖4B柱色谱、羟基磷灰石柱色谱和葡聚糖G - 200凝胶过滤。胎盘酶和肾脏酶的最终回收率分别为22.0%和20.8%。聚丙烯酰胺凝胶电泳、分析等电聚焦、免疫电泳和超速离心表明两种纯化酶均具有均一性。平衡超速离心显示胎盘酶的分子量为193000，肾脏酶的分子量为211000。在十二烷基硫酸盐/聚丙烯酰胺凝胶上进行变性条件下的酶电泳表明，每种酶都是由两个相同亚基组成的二聚体。胎盘酶和肾脏酶在4℃时的等电点分别为4.20和4.32。两种膜结合芳基酰胺酶的氨基酸组成相似。碳水化合物占胎盘酶的18.2%，占肾脏酶的17.4%。纯化的胎盘酶的比活性为60.1 μmol min⁻¹（mg蛋白质）⁻¹，肾脏酶的比活性为96.4 μmol min⁻¹（mg蛋白质）⁻¹。胎盘酶和肾脏酶的Kcat值分别为187.8 s⁻¹和328.6 s⁻¹。胎盘酶对L - 丙氨酰 - β - 萘酰胺的水解系数（Kcat/Km）为2159 mM⁻¹ s⁻¹，肾脏酶的水解系数为3778 mM⁻¹ s⁻¹。针对胎盘和肾脏膜结合芳基酰胺酶的兔抗体抑制相应抗原的活性，但抑制作用从未完全。来自胎盘、肾脏和肾细胞癌的三种膜结合芳基酰胺酶在免疫化学上无法区分。