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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Adh 3 - 1-和野生小鼠施用不同剂量的乙醇后血液乙醇浓度的分析表明，III类ADH剂量依赖性地促进体内酒精代谢，尽管III类ADH在血液水平上对乙醇显示出较差的体外活性。这种ADH也有助于保持γ值恒定，即使在高剂量的乙醇。我们用一种新研制的多功能显微光谱仪研究了组织细胞环境中Class III ADH活性的调节，发现Class III ADH在模拟组织细胞内环境的疏水环境中，包括脂质体的膜结构，被乙醇激活。此外，III类ADH在体外和体内的平滑肌细胞中通过由于膜损伤引起的疏水性增加而被诱导，在体内的肝脏中被诱导。 ...更多信息改变大剂量乙醇给药。这些结果表明，III类ADH提高其在体内的酒精代谢的贡献，激活和诱导在大剂量的乙醇，剂量增加细胞的疏水性，由于肝损伤，为了探讨激活III类ADH的机制，增加溶液的疏水性，活性和蛋白质结构之间的关系进行了研究，使用重组ADH在疏水溶液中。圆二色性（CD）光谱法显示，在疏水溶液中，III类ADH，但不是I类ADH被激活的I类和III类的二级结构没有显着变化。因此，我们得出结论，溶液疏水性的变化诱导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。在疏水条件下，构成Class III口袋的亲水性氨基酸折叠，使口袋缩小，从而提高酶对小分子乙醇的亲和力，提高酶对血液中乙醇的活性。克隆了小鼠Class III ADH基因的基因组DNA片段，包括全部9个外显子、8个内含子、14 kb的5'非翻译区和10 kb的3'非翻译区，并对该基因的全长进行了DNA序列分析。在约2kb的5' UTR区域内发现了一些罕见的转录因子基序序列，如XRE、STAT和SRY。这表明小鼠Class III ADH的表达受毒性化合物、激素和性别决定因子的调节，因此，Class III ADH的活性受其在底物结合口袋中的结构变化的调节，所述结构变化是由于溶液疏水性的改变和基因的转录调节，从而有助于体内酒精代谢以及其他生理代谢。Ⅲ类抗利尿激素对酒精代谢贡献的增加可能与酒精中毒时组织损害的发生有关。少