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

本研究分析了NADH-细胞色素b_5还原酶的基因结构和表达调控。由于我们已经克隆并测定了人肝脏和胎盘的NADH-细胞色素b_5还原酶的cDNA，我们用该cDNA作为探针分析了人基因组DNA中的NADH-细胞色素b_5还原酶。在基因组DNA中共发现9个外显子，超过30 K碱基对。该酶的膜存在形式的切割位点位于第2外显子的中心附近。该酶的遗传缺陷可分为3种类型。它们是红细胞型（I型）、全身型（II型）和血细胞型（III型）。本研究首先分析了该患者组织的基因结构，发现在患者组织中存在一个由Ser-127的密码子TCA到Pro的密码子CCA的点突变。通过定点突变的方法将突变基因导入到该酶的cDNA中，并在大肠杆菌中表达，纯化突变酶并对其进行鉴定。突变酶Ser-127-> Pro具有异常的吸收光谱、荧光光谱和圆二色谱。NADH的表观Km值比野生型酶的表观Km值高约10倍，并且Vmax是野生型酶的约30%。在Ser-127->Ala突变体中也观察到类似的性质。因此，Ser-127的OH-基团在维持NADH结合位点的结构中起重要作用。

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.