| Project/Area Number |
11470120
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Legal medicine
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| Research Institution | NIPPON MEDICAL SCHOOL |
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Principal Investigator |
HASEBA Takeshi NIPPON MED. SCH., LEGAL MED., ASSIST. PROF., 医学部, 講師 (50156329)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAMOTO Isao KANAGAWA DENT. SCH., LAGAL MED., ASSIST., 歯学部, 助手 (30277917)
KAMEYAMA Kouji NIPPON MED. SCH., PATHOL., ASSIST., 医学部, 助手 (60150736)
MASHIMO Keiko NIPPON MED. SCH., LEGAL MED., ASSIST., 医学部, 助手 (90113000)
KUROSAWA Norio SOKA UNIV., ENGINEERING ASSOC., PROF., 工学部, 助教授 (30234602)
SHIMIZU Akio SOKA UNIV., ENGINEERING ASSOC., PROF., 工学部, 助教授 (20235641)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥13,900,000 (Direct Cost: ¥13,900,000)
Fiscal Year 2001: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2000: ¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 1999: ¥6,400,000 (Direct Cost: ¥6,400,000)
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| Keywords | CLASS III ALCOHOL DFHYDROGENASE / KNOCKOUT MOUSE / ALCOHOL METABOLISM / TISSUE DAMAGES / MICROSPECTROMETRY / ENZYME REGULATION / GENOMIC DNA ANALYSIS / CLASS I ALCOHOL DEHYDROGENASE / リコンビナントADH / 活性・構造相関 / 顕微分光酵素活性測定 / Class III ADH遺伝子 / ADH強発現株細胞 / Class III ADH遺伝子クローニング / マウス / 肝ADH酵素量 / ADH大量発現系 |
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| Research Abstract |
Analyses of blood ethanol concentrations after administration of ethanol at various doses to Adh3-l- and Wild mice demonstrated that Class III ADH contributes dose-dependently to alcohol metabolism in vivo, although the Class III ADH shows a poor activity m vitro for ethanol at blood levels. This ADH also contributes to keep the γ value constant even at high doses of ethanol. By such contributions of this ADH to alcohol pharmacokinetics, Class III ADH was shown to alleviate acute alcoholic intoxication.
We investigated the regulation of Class III ADH activity in cellular environment of tissues, by using a newly developed multifunctional microspectrometer and found that Class III ADH was activated for ethanol in hydrophobic environments including membrane structure of liposome, which mimicked intracellular environments of tissues. Moreover, Class III ADH was induced m the smooth muscle cells in vitro and in vivo by increased hydrophobicity due to membrane damages and in the liver m vivo
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alter administration of ethanol at large doses. These results imply that Class III ADH elevates its contribution to alcohol metabolism in vivo by both activation and induction at large doses of ethanol, which doses increase cellular hydrophobicity due to liver damage.
In order to investigate the mechanism of activation of Class III ADH by increased solution hydrophobicity, the relation between the activity and protein structure in hydrophobic solutions was studied using recombinant ADHs. Circular dichroism (CD) spectrometry showed no significant changes in the secondary structure of both Class I and III in the hydrophobic solution where Class III ADH, but not Class I ADH, is activated. Therefore, we concluded that a change of solution hydrophobicity induces structural fluctuation in the active site of Class III ADH, because the substrate-binding pocket of human Class III ADH is known to be more hydrophilic and larger than that of Class I ADH. In hydrophobic conditions hydrophilic amino acid constructing the pocket of Class III collapse to reduce the pocket size, which may raise the affinity of the enzyme for ethanol of a small molecule and increase the enzyme activity for ethanol of blood levels.
In addition, we investigated a translational regulation of Class III ADH at gene level. We cloned genomic DNA fragments of mouse Class III ADH gene including all 9 exons, 8 introns, 14kb of 5' UTR and 10 kb of 3' UTR and completed the DNA sequence analysis over the whole length of the gene. Several rare sequences such as XRE, STAT and SRY were found as motif sequences for transcriptional factors in the region of about 2 kb 5' UTR. This suggests that the expression of mouse Class III ADH is regulated by toxic compounds, hormones and sex-determining factors.
Thus, the activity of Class III ADH is regulated by its structural changes in the substrate-binding pocket due to altered solution hydrophobicity and the transcriptional regulation of the gene so as to contribute to alcohol metabolism in vivo as well as other physiological metabolisms. The increase of contribution of Class III ADH to alcohol metabolism may relate to development of tissue damages in alcoholism. Less
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