1989 Fiscal Year Final Research Report Summary
Gene structure of NADH-cytochrome b_5 reductase and regulation of expression
| Project/Area Number |
63570121
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Medical College of Oita |
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Principal Investigator |
YUBISUI Toshitsugu Medical College of Oita, School of Medicine, Assistant Professor, 医学部, 助教授 (00019564)
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| Co-Investigator(Kenkyū-buntansha) |
SHIRABE Komei Medical College of Oita, School of Medicine, Instructor, 医学部, 助手 (50179058)
YOSHIDA Satoshi Medical College of Oita, School of Medicine, Assistant Professor, 医学部, 助教授 (50158440)
TAKESHITA Masazumi Medical College of Oita, School of Medicine, Professor, 医学部, 教授 (50019551)
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| Project Period (FY) |
1988 – 1989
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| Keywords | NADH-cytochrome b_5 reductase / Gene structure / Hereditary deficiency / Point mutation / Ser-127->Pro mutant / Mutant enzyme |
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| Research Abstract |
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.
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Research Products
(13 results)
