Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog.
Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog.
复制标题
使用固定化过渡态类似物通过亲和层析纯化大鼠肝脏中的鸟氨酸转氨甲酰酶。
DOI：
10.1016/0003-2697(80)90045-7
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发表时间：
1980
影响因子：
2.9
通讯作者：
G. Howlett
中科院分区：
文献类型：
--
作者：
N. Hoogenraad;T. M. Sutherland;G. Howlett
从大鼠肝脏中将鸟氨酸转氨甲酰酶（EC 2.1.3.3）纯化至均一。该方法的基础是在亲和柱上对大鼠肝脏匀浆的高速上清液部分进行色谱分析，该亲和柱由鸟氨酸转氨甲酰酶的过渡态类似物δ-N-（膦酰基乙酰基）-l-鸟氨酸组成，通过α-氨基固定在环氧活化的Sepharose 6 B上。使用底物氨基甲酰磷酸的梯度从柱上洗脱酶，并通过凝胶过滤进一步纯化。该酶在pH 8.5，37°C下以250至260 μmol min− 1 mg − 1的恒定比活性洗脱，并且在十二烷基硫酸钠凝胶电泳上不含污染蛋白。通过离心（98，000）和在十二烷基硫酸钠存在下通过凝胶电泳（35，300）测定纯化酶的分子量，表明来自大鼠肝脏的酶是三聚体。该酶在pH 7.4下表现出常规的Michaelis-Menten动力学，并且在这方面不同于通过其它方法制备的酶。
Ornithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supernatant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of ornithine transcarbamylase, δ-N-(phosphonacetyl)-l-ornithine, immobilized on epoxy-activated Sepharose 6B through the α-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 μmol min−1mg−1at pH 8.5, 37°C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods.