CLONING AND FUNCTIONAL EXPRESSION OF A SOLUBLE FORM OF KYNURENINE ALPHA-AMINOADIPATE AMINOTRANSFERASE FROM RAT-KIDNEY
CLONING AND FUNCTIONAL EXPRESSION OF A SOLUBLE FORM OF KYNURENINE ALPHA-AMINOADIPATE AMINOTRANSFERASE FROM RAT-KIDNEY
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DOI：
10.1074/jbc.270.49.29330
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发表时间：
1995-12-08
影响因子：
4.8
通讯作者：
CESURA, AM
中科院分区：
文献类型：
--
作者：
BUCHLI, R;ALBERATIGIANI, D;CESURA, AM
几种具有犬尿氨酸氨基转移酶（KAT）活性的氨基转移酶能够将L-犬尿氨酸转化为犬尿烯酸，犬尿烯酸是一种推定的内源性多巴胺能神经传递调节剂。在大鼠中，已发现所述KAT同种型之一对应于谷氨酰胺转氨酶K。此外，大鼠肾脏α-氨基己二酸氨基转移酶（AadAT）也显示KAT活性。在本报告中，我们描述了编码大鼠这种氨基转移酶同工酶可溶性形式（KAT/AadAT）的cDNA克隆的分离。从大鼠肾脏KAT/AadAT胰蛋白酶肽的氨基酸序列设计简并寡核苷酸，用作大鼠肾脏RNA的逆转录-聚合酶链反应的引物。将得到的聚合酶链反应片段用于筛选大鼠肾cDNA文库并分离编码KAT/AadAT的cDNA克隆。组合DNA序列的分析表明存在编码425个氨基酸残基的可溶性蛋白的单个1275碱基对开放阅读框。KAT/AadAT似乎在吡哆醛5 '-磷酸结合结构域中与天冬氨酸氨基转移酶结构同源。RNA印迹分析大鼠组织，包括脑，揭示了一个单一的种类的KAT/AadAT mRNA的类似2.1激酶。用KAT/AadAT cDNA转染的HEK-293细胞表现出HAT和AadAT活性，其酶性质类似于大鼠天然蛋白质所报道的那些。
Several aminotransferases with kynurenine aminotransferase (KAT) activity are able to convert L-kynurenine into kynurenic acid, a putative endogenous modulator of glutamatergic neurotransmission. In the rat, one of the described KAT isoforms has been found to correspond to glutamine transaminase K. In addition, rat kidney alpha-aminoadipate aminotransferase (AadAT) also shows KAT activity. In this report, we describe the isolation of a cDNA clone encoding the soluble form of this aminotransferase isoenzyme from rat (KAT/AadAT). Degenerate oligonucleotides were designed from the amino acid sequences of rat kidney KAT/AadAT tryptic peptides for use as primers for reverse transcription-polymerase chain reaction of rat kidney RNA. The resulting polymerase chain reaction fragment was used to screen a rat kidney cDNA library and to isolate a cDNA clone encoding KAT/AadAT. Analysis of the combined DNA sequences indicated the presence of a single 1275-base pair open reading frame coding for a soluble protein of 425 amino acid residues. KAT/AadAT appears to be structurally homologous to aspartate aminotransferase in the pyridoxal 5'-phosphate binding domain. RNA blot analysis of rat tissues, including brain, revealed a single species of KAT/AadAT mRNA of similar to 2.1 kilobases. HEK-293 cells transfected with the KAT/AadAT cDNA exhibited both HAT and AadAT activities with enzymatic properties similar to those reported for the rat native protein.