Expression of functional human glutaminase in baculovirus system:: Affinity purification, kinetic and molecular characterization
Expression of functional human glutaminase in baculovirus system:: Affinity purification, kinetic and molecular characterization
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DOI：
10.1016/j.biocel.2006.12.002
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发表时间：
2007-01-01
影响因子：
4
通讯作者：
Marquez, Javier
中科院分区：
文献类型：
--
作者：
Campos-Sandoval, Jose A.;de la Oliva, Amada R. Lopez;Marquez, Javier
谷氨酰胺酶催化谷氨酰胺的水解，产生化学计量的谷氨酸盐和铵离子。在哺乳动物中，有两种不同的基因编码转氨酶，称为肝（L）和肾（K）型。杆状病毒表达的人L型亚型在Sf 9昆虫细胞中产生功能性重组酶。基于其与PDZ蛋白的特异性相互作用，开发了一种新的亲和层析方法用于纯化。动力学常数被确定为纯化的人同工酶，这表明谷氨酰胺的变构行为，希尔指数为2.7和S-0.5值为32和64 mM的高和低的P-i浓度，分别。而蛋白质表现出低的P-1依赖性典型的L-型精氨酸酶，该酶出乎意料地抑制谷氨酸，动力学特征排除K-型同工酶，并轻微激活氨，不像经典的肝酶，显示出绝对依赖于氨。亚细胞分级分离证明重组人谷氨酰胺酶靶向线粒体和细胞核，并且在这两个位置中蛋白质都具有催化活性。这是第一个报告的功能性L型哺乳动物转氨酶的表达。该研究为重组酶的亲和纯化提供了一种简便、有效的方法。此外，这些数据意味着，这种人类酶可能代表了一种新的同工酶不同于经典的肾脏和肝脏同工酶。(c)2006爱思唯尔有限公司保留所有权利。
Glutaminase catalyzes the hydrolysis of glutamine yielding stoichiometric amounts of glutamate plus ammonium ions. In mammals, there are two different genes encoding for glutaminase, known as liver (L) and kidney (K) types. The human L-type isoform expressed in baculovirus yielded functional recombinant enzyme in Sf9 insect cells. A novel affinity chromatography method, based on its specific interaction with a PDZ protein, was developed for purification. Kinetic constants were determined for the purified human isozyme, which showed an allosteric behaviour for glutamine, with a Hill index of 2.7 and S-0.5 values of 32 and 64 mM for high and low P-i concentrations, respectively. Whereas the protein showed a low P-i dependence typical for L-type glutaminases, the enzyme was unexpectedly inhibited by glutamate, a kinetic characteristic exclusive of K-type isozymes, and was slightly activated by ammonia, unlike the classical liver enzymes which show an absolute dependence on ammonia. Subcellular fractionation demonstrates that recombinant human glutaminase was targeted to both mitochondria and nucleus, and in both locations the protein was catalytically active. This is the first report of the expression of a functional L-type mammalian glutaminase enzyme. The study also provides a simple and efficient method for affinity purification of the recombinant enzyme. Moreover, the data imply that this human enzyme may represent a new isoform different from classical kidney and liver isozymes. (c) 2006 Elsevier Ltd. All rights reserved.