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-

对adh3 - 1和野生小鼠给予不同剂量乙醇后的血液乙醇浓度分析表明，III类ADH在体内对酒精代谢有剂量依赖性，尽管III类ADH在体外对血液水平的乙醇的活性较差。这种ADH也有助于保持γ值恒定，即使在高剂量的乙醇。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。我们利用新开发的多功能显微光谱仪研究了III类ADH在组织细胞环境中的活性调控，发现III类ADH在疏水环境中被乙醇激活，包括脂质体的膜结构，这种环境模拟了组织的细胞内环境。此外，III类ADH在体外和体内通过膜损伤引起的疏水性增加在平滑肌细胞和肝脏中诱导。这些结果表明，III类ADH通过大剂量乙醇的激活和诱导，提高了其对体内酒精代谢的贡献，而乙醇的剂量增加了肝损伤引起的细胞疏水性。为了探讨III类ADH通过提高溶液疏水性而活化的机制，利用重组ADH研究了其活性与疏水性溶液中蛋白质结构的关系。圆二色性（CD）光谱分析显示，在激活III类ADH而非I类ADH的疏水溶液中，I类和III类ADH的二级结构没有显著变化。因此，我们得出结论，溶液疏水性的变化导致了III类ADH活性位点的结构波动，因为已知人类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，构建III类口袋的亲水性氨基酸坍塌，使口袋变小，这可能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。此外，我们还研究了III类ADH在基因水平上的翻译调控。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XRE、STAT和SRY等罕见序列作为转录因子的基序序列。提示小鼠III类ADH的表达受毒性化合物、激素和性别决定因素的调控。因此，III类ADH的活性受溶液疏水性改变导致底物结合袋结构变化和基因转录调控的影响，从而参与体内酒精代谢及其他生理代谢。III类ADH对酒精代谢贡献的增加可能与酒精中毒组织损伤的发展有关。少

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