ROLE OF CLASS III ALCOHOL DEHYDROGENASE (ADH3), A HOUSEKEEPING ENZYME FOR CYTOTOXICITY, IN ALCOHOL METABOLISM -IN VIVO STUDY USING KNOCKOUT MICE AND INVITRO STUDY ON ENZYME REGULATION-

III 类酒精脱氢酶 (ADH3)（一种细胞毒性管家酶）在酒精代谢中的作用 - 使用敲除小鼠的体内研究和酶调节的体外研究 -

基本信息

批准号：
11470120
负责人：
HASEBA Takeshi
金额：
$ 8.9万
依托单位：
NIPPON MEDICAL SCHOOL
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

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 … More 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

对 Adh3-l- 和 Wild 小鼠给予不同剂量的乙醇后血液乙醇浓度的分析表明，III 类 ADH 对体内酒精代谢的贡献呈剂量依赖性，尽管 III 类 ADH 在体外对血液中的乙醇表现出较差的活性即使在高剂量的乙醇下，这种 ADH 也有助于保持 γ 值恒定。通过这种 ADH 对酒精药代动力学的贡献，III 类 ADH 被证明可以缓解急性酒精中毒。我们研究了其调节作用。通过使用新开发的多功能微型光谱仪，研究人员对组织细胞环境中的 III 类 ADH 活性进行了研究，发现在模拟组织细胞内环境的疏水环境（包括脂质体的膜结构）中，III 类 ADH 被乙醇激活。通过膜损伤和肝脏体内的疏水性增加，在体外和体内诱导平滑肌细胞……更多改变大剂量乙醇的给药这些结果表明，III 类 ADH 通过以下方式提高其对体内酒精代谢的贡献。在大剂量乙醇下同时激活和诱导，由于肝损伤，该剂量增加了细胞疏水性。为了研究通过增加溶液疏水性来激活III类ADH的机制，研究了疏水溶液中活性与蛋白质结构之间的关系使用重组 ADH 进行圆二色性 (CD) 光谱分析显示，在 III 类 ADH（而非 I 类 ADH）被激活的疏水溶液中，I 类和 III 类的二级结构没有显着变化。溶液疏水性的变化会引起 III 类 ADH 活性位点的结构波动，因为已知人 III 类 ADH 的底物结合口袋比 I 类 ADH 更亲水且更大。 III类ADH的口袋塌陷以减小口袋尺寸，这可能会提高酶对小分子乙醇的亲和力，并增加血液中乙醇的酶活性。此外，我们研究了III类ADH的翻译调控基因我们克隆了小鼠III类ADH基因的基因组DNA片段，包括全部9个外显子、8个内含子、14kb的5'UTR和10kb的3'UTR，并完成了该基因全长的DNA序列分析。在约 2 kb 5' UTR 区域发现了诸如 XRE、STAT 和 SRY 等转录因子的基序序列，这表明小鼠 III 类 ADH 的表达受到调节。因此，III类ADH的活性通过其底物结合袋中由于溶液疏水性改变而发生的结构变化以及基因的转录调节来调节，从而有助于乙醇代谢III 类 ADH 对酒精代谢的贡献增加可能与酒精中毒时组织损伤的发生有关。