Brain dysfunction and lipid metabolism in hereditary methemoglobinenia generalized type

为了评估人红细胞细胞色素b5血红素周围羧基残基的作用，我们利用大肠杆菌细胞色素b5的定点诱变和表达系统，制备并鉴定了由Ala取代Glu41、Glu42、Asp57、Glu63、Asp70和Glu73的细胞色素b5突变体。野生型nadh -细胞色素b5还原酶对Glu42Ala细胞色素b5和Asp70Ala细胞色素b5的表观Km值分别比野生型细胞色素b5高约3倍和6倍。相反，这些突变体的kcat值没有受到显著影响。此外，对细胞色素b5和b5Rs突变体组合的动力学研究表明，在反应中，细胞色素b5的Glu42和Asp70可能分别与nadh -细胞色素b5还原酶的Lys125和Lys41相互作用。在红细胞、肝脏、脑和HL-60细胞中发现了一种新的人b5还原酶（H.b5R） mRNA。与先前表征的H.b5R mRNA相比，它至少有两个起始位点，并且包含一个替代的非编码第一外显子。这种mRNA在红细胞中的转录水平相对高于肝脏和脑细胞。该mRNA的第一个外显子与大鼠红细胞特异性b5R mRNA的第一个外显子及其直接下游内含子序列具有62%的同源性，而该H.b5R mRNA的假设启动子具有家养基因的特征，类似于大鼠普遍存在的一种新型b5R mRNA。这些结果可能对理解H.b5R生成的调控机制具有重要意义。

To evaluate the role of carboxyl residues surrounding heme of human erythrocyte cytochrome b5, we prepared and characterized the cytochrome b5 mutants in which Glu41, Glu42, Asp57, Glu63, Asp70, and Glu73 were replaced by Ala, utilizing site-directed mutagenesis and expression system for cytochrome b5 in Escherichia coli. Apparent Km values of the wild type NADH-cytochrome b5 reductase for Glu42Ala cytochrome b5 and Asp70Ala cytochrome b5 were approximately three-fold and six-fold higher than that for the wild type cytochrome b5, respectively. In contrast, the kcat values for those mutants were not remarkably affected. Furthermore, kinetic studies on combinations of the cytochrome b5 and b5Rs mutants suggested the possible interaction between Glu42 and Asp70 of cytochrome b5 and Lys125 and Lys41 of NADH-cytochrome b5 reductase, respectively, in the reaction.A new type of human b5 reductase (H.b5R) mRNA was found from erythrocyte, liver, brain and HL-60 cells. It has at least two initiate sites and contains an alternative non-coding first exon in comparision with the H.b5R mRNA characterized previously. The transcription level for this mRNA is relatively higher in erythrocyte than that in liver and in brain cells. The first exon of this mRNA has 62% of homology with the first exon and its immediate downstream intron sequnces of a rat erythrocyte-specific b5R mRNA,whereas, the putative promoter of this H.b5R mRNA possesses features of house keeping gene, similar to one of the ubiquitous novel b5R mRNAs of rat. These results may be important in understanding the regulation mechanism of H.b5R generation.