|Budget Amount *help
¥5,100,000 (Direct Cost : ¥5,100,000)
Fiscal Year 1997 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1996 : ¥3,500,000 (Direct Cost : ¥3,500,000)
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.