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Gene structure of NADH-cytochrome b_5 reductase and regulation of expression

NADH-细胞色素b_5还原酶的基因结构及表达调控

基本信息

批准号：
63570121
负责人：
YUBISUI Toshitsugu
金额：
$ 1.41万
依托单位：
Medical College of Oita
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

In this study. the gene structure and regulation of expression of NADH-cytochrome b_5 reductase has been analyzed. As we have already cloned and determined the cDNA for human liver and placenta NADH-cytochrome b_5 reductase, we analyzed genomic DNA for the NADH-cytochrome b_5 reductase using the cDNA as the probe. Total of 9 exons were found in the genomic DNA over 30K base-pairs. The cleavage site of the membrane-found form of the enzyme was found at around the center in the 2nd exon.Hereditary deficiency of this enzyme is classified in 3 types. Those are erythrocyte-type (Type I), generalized type (Type II) and blood cell-type (Type III). Gene structure of tissues from the generalized patient was analyzed at first in this study, and a point mutation of the codon TCA for Ser-127 to CCA for Pro was found to expressed in the patient tissues. This mutation was introduced into the cDNA of this enzyme by the sitedirected mutagenesis, and the mutant cDNA was expressed in Escherichia coli, and mutant enzyme was purified to characterize. The mutant enzyme, Ser-127-> Pro, showed abnormal absorption, fluorescence and circular dichroism spectra. An apparent Km value for NADH is about 10 times higher than that of the wild-type enzyme, and Vmax is about 30% of that of the wild-type enzyme. Similar properties were also observed with the Ser-127->Ala mutant. Therefore, OH-group of Ser-127 is concluded to play an important role to maintain the structure of NADH-binding site.

在这项研究中。分析了nadh -细胞色素b_5还原酶的基因结构及其表达调控。由于我们已经克隆并测定了人肝脏和胎盘nadh -细胞色素b_5还原酶的cDNA，我们以nadh -细胞色素b_5还原酶为探针，分析了nadh -细胞色素b_5还原酶的基因组DNA。在基因组DNA中共发现9个超过30K碱基对的外显子。膜发现形式的酶的切割位点位于第2外显子的中心附近。这种酶的遗传性缺乏可分为三种类型。它们是红细胞型（I型），广义型（II型）和血细胞型（III型）。本研究首先分析了广泛性患者组织的基因结构，发现Ser-127密码子TCA到Pro密码子CCA的点突变在患者组织中表达。通过定点诱变将该突变引入该酶的cDNA中，在大肠杆菌中表达，并纯化突变酶进行鉴定。突变酶Ser-127-> Pro表现出异常的吸收、荧光和圆二色光谱。NADH的表观Km值约为野生型酶的10倍，Vmax约为野生型酶的30%。在Ser-127->Ala突变体中也观察到类似的特性。因此，Ser-127的oh基团对维持nadh结合位点的结构起重要作用。