CHARACTERIZATION OF A CDNA CLONE FOR HUMAN NAD(+)-SPECIFIC ISOCITRATE DEHYDROGENASE ALPHA-SUBUNIT AND STRUCTURAL COMPARISON WITH ITS ISOENZYMES FROM DIFFERENT SPECIES
CHARACTERIZATION OF A CDNA CLONE FOR HUMAN NAD(+)-SPECIFIC ISOCITRATE DEHYDROGENASE ALPHA-SUBUNIT AND STRUCTURAL COMPARISON WITH ITS ISOENZYMES FROM DIFFERENT SPECIES
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DOI：
10.1042/bj3080063
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发表时间：
1995-05-15
影响因子：
4.1
通讯作者：
HUH, TL
中科院分区：
文献类型：
--
作者：
KIM, YO;OH, IU;HUH, TL
使用基于猪胰蛋白酶肽序列合成的寡核苷酸引物，通过PCR扩增编码人NAD（+）特异性异柠檬酸脱氢酶α-亚基（H-IDH α）的0.6kb cDNA片段[Huang和Colman（1990）Biochemistry 29，8266-8273]。以扩增的cDNA为探针，从人心脏λ gt 11 cDNA文库中克隆了IDH α的cDNA克隆，最大的cDNA克隆（2628 bp）的蛋白质序列为366个氨基酸（39 591 Dal）的前体蛋白和339个氨基酸（36640 Dal）的成熟蛋白。推导的H-IDH α蛋白序列与猪IDH α蛋白的部分肽段序列高度相似，与酵母NAD（+）特异性IDH 2、酵母NAD（+）特异性IDH 1和猴NAD（+）特异性IDH γ亚基（IDH γ）的同源性分别为55%、43%和44%。然而，它与来自大肠杆菌和牛线粒体的NADP（+）特异性IDH的相似性较小（约3.0%）。这些结果表明，IDH α的结构与酵母酶的催化亚基IDH 2的结构非常相似。H-IDH α蛋白的结构分析表明，与异柠檬酸、Mg ~（2+）和NAD结合的氨基酸高度保守，与ATP和ADP结合的氨基酸也具有两个保守的基序，但没有识别出典型的Ca ~（2+）结合基序。不寻常的五-（ATTTA）和三-（TAA或ATT）核苷酸，分别被认为是相互作用的RNA结合蛋白和附近的核酸内切酶切割位点，经常被识别在其3'非翻译区，这表明一个额外的方法调节这种酶的可能性。Northern印迹分析表明，在培养的HeLa细胞中存在一个mRNA转录本（2.8kb）。基因组DNA Southern-blot分析表明，IDH α基因与其他IDH同工酶的基因不密切相关，IDH α似乎由单个基因编码。
A 0.6 kb cDNA fragment encoding the human NAD(+)-specific isocitrate dehydrogenase alpha-subunit (H-IDH alpha) was amplified by PCR using oligonucleotide primers synthesized on the basis of pig tryptic peptide sequences [Huang and Colman (1990) Biochemistry 29, 8266-8273]. With the amplified cDNA as a probe, cDNA clones for IDH alpha were isolated from a human heart lambda gt11 cDNA library, The deduced protein sequence of the largest cDNA clone (2628 bp) rendered a precursor protein of 366 amino acids (39 591 Dal and a mature protein of 339 amino acids (36640 Dal. The deduced H-IDH alpha protein sequence is highly similar to the partial peptide sequences of the pig enzyme, It is 55, 43 and 44% identical with yeast NAD(+)-specific IDH2, yeast NAD(+)-specific IDH1 and monkey NAD(+)-specific IDH gamma-subunit (IDH gamma) respectively. However, it has less similarity (about 3.0%) to NADP(+)-specific IDH from Escherichia coli and bovine mitochondria. These results indicate that the structure of IDH alpha closely resembles that of IDH2, the catalytic subunit of the yeast enzyme, Structural analysis of the deduced H-IDH alpha protein revealed that the amino acids responsible for the binding of isocitrate, Mg2+ and NAD are highly conserved, It also has two conserved motifs for the binding sites of ATP and ADP, but a canonical Ca2+-binding motif was not recognized. Unusual penta- (ATTTA) and tri-(TAA or ATT) nucleotides which are respectively believed to interact with RNA-binding proteins and be near the endonuclease cleavage sites were frequently recognized in its 3' untranslated region, indicating the possibility of an additional method of regulation of this enzyme. Northern-blot analysis suggests that one mRNA transcript (2.8 kb) exists in cultured HeLa cells. Genomic DNA Southern-blot analysis indicates that the IDH alpha gene is not closely related to that of the other IDH isoenzymes, and IDH alpha appears to be encoded by a single gene.