Kalousek F, François B, Rosenberg L E
J Biol Chem. 1978 Jun 10;253(11):3939-44.
We report experiments describing the isolation and characterization of ornithine transcarbamylase from normal human liver. Our preparative procedure employs initial centrifugation and heat steps, intermediate batch-wise adsorption and desorption from ion exchange resins and column chromatographic elution from hydroxylapatite, and final purification by gel filtration chromatography and glycerol density gradient centrifugation. The enzyme, purified 580-fold in this way, is homogeneous as judged by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Human ornithine transcarbamylase has a molecular weight of 114,000 and is a trimer of identical 38,000 molecular weight subunits. It focuses at pH 6.8 as a single band on polyacrylamide gel, has a COOH-terminal phenylalanine, an NH2-terminal glycine, an apparent Km for L-ornithine of 0.4 mM and for carbamyl phosphate of 0.16 mM, and a pH optimum of 7.7. The enzyme is quite stable over a temperature range from -50 degrees to +60 degrees C and over the pH range from 5.8 to 8.2. The quaternary structure and amino acid composition of the human enzyme are very similar to those of its bovine homologue.
我们报告了有关从正常人肝脏中分离和鉴定鸟氨酸转氨甲酰酶的实验。我们的制备方法包括初始离心和加热步骤、离子交换树脂的中间分批吸附和解吸以及羟基磷灰石柱色谱洗脱，最后通过凝胶过滤色谱和甘油密度梯度离心进行纯化。通过这种方式纯化了580倍的酶，根据天然和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳判断为均一。人鸟氨酸转氨甲酰酶的分子量为114,000，是由相同的38,000分子量亚基组成的三聚体。它在聚丙烯酰胺凝胶上作为单一带聚焦于pH 6.8，具有羧基末端苯丙氨酸、氨基末端甘氨酸、对L-鸟氨酸的表观Km为0.4 mM，对氨甲酰磷酸的表观Km为0.16 mM，最适pH为7.7。该酶在-50℃至+60℃的温度范围内以及pH 5.8至8.2的范围内相当稳定。人酶的四级结构和氨基酸组成与其牛同源物非常相似。
