Isolation, characterization and expression of a human brain mitochondrial glutaminase cDNA
Isolation, characterization and expression of a human brain mitochondrial glutaminase cDNA
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DOI：
10.1016/s0169-328x(99)00331-9
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发表时间：
2000-03-10
期刊：
影响因子：
--
通讯作者：
Curthoys, NP
中科院分区：
文献类型：
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作者：
Holcomb, T;Taylor, L;Curthoys, NP
对编码全长人脑谷氨酰胺酶（GA）cDNA的重叠部分的各种cDNA进行克隆和测序。hGA的总体核苷酸序列与大鼠肾型GA cDNA（77.4%）和编码5.0-kb猪CIA mRNA的cDNA的已知部分（81.1%）具有非常高的同一性。同一性在氨基酸水平上甚至更显著，特别是在C-末端一半，其中三种蛋白质共享99.7%的序列同一性。hGA cDNA编码73，427-Da蛋白，其含有N-末端线粒体靶向信号并保留大鼠肾线粒体转氨酶的胞质前体表征的主要蛋白水解切割位点。通过使用独特的限制性位点组装整个编码区并克隆到杆状病毒中。用重组病毒感染的sf 9细胞表达高水平的适当加工的和活性的转氨酶。因此，分离的hGA cDNA的表达应该提供一种纯化大量线粒体谷氨酰胺酶的方法，所述线粒体谷氨酰胺酶是催化谷氨酰胺代谢和重要的兴奋性和抑制性神经递质的合成中的关键反应的蛋白质。(C)2000 Elsevier Science B. V.保留所有权利。
Various cDNAs that encode overlapping portions of the full-length human brain glutaminase (GA) cDNA were cloned and sequenced. The overall nucleotide sequence of hGA has a very high degree of identity with that of the rat kidney-type GA cDNA (77.4%) and the known portion of the cDNA that encodes the 5.0-kb porcine CIA mRNA (81.1%). The identity is even more remarkable at the amino acid level, particularly in the C-terminal half where the three proteins share a 99.7% sequence identity. The hGA cDNA encodes a 73,427-Da protein that contains an N-terminal mitochondrial targeting signal and retains the primary proteolytic cleavage site characterized fur the cytosolic precursor of the rat renal mitochondrial glutaminase. The entire coding region was assembled through the use of unique restriction sites and cloned into a baculovirus. Sf9 cells infected with the recombinant virus express high levels of properly processed and active glutaminase. Thus, expression of the isolated hGA cDNA should provide a means to purify large amounts of the mitochondrial glutaminase, a protein that catalyzes a key reaction in the metabolism of glutamine and the synthesis of important excitatory and inhibitory neurotransmitters. (C) 2000 Elsevier Science B.V. All rights reserved.