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-L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型ADH对体内乙醇代谢的贡献具有剂量依赖性，尽管III型ADH在体外对血液水平的乙醇表现出较低的活性。这种ADH还有助于即使在高剂量乙醇下也保持γ值不变。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组织细胞环境中III类ADH活性的调节，发现在包括脂质体膜结构在内的疏水环境中，III类ADH被激活为乙醇，模拟组织内的细胞内环境。此外，在体外和体内，由于膜损伤导致疏水性增加，在体外和体内的血管平滑肌细胞和体内的肝脏…都诱导了III型adh的产生。更多的是改变了大剂量乙醇的使用。这些结果表明，在体内，III类ADH通过在大剂量乙醇中的激活和诱导而提高其对酒精代谢的贡献，从而增加肝脏损伤所致的细胞疏水性。为了探讨溶液疏水性增加而激活III类ADH的机制，利用重组ADHS研究了疏水溶液中ADH活性与蛋白质结构的关系。圆二色谱(CD)表明，在被激活的III类ADH而不是I类ADH的疏水溶液中，I类和III类的二级结构没有显著变化。因此，我们得出结论，溶液疏水性的改变会导致第III类ADH活性部位的结构变化，因为已知人类第III类ADH的底物结合口袋比第I类ADH更亲水且更大。在疏水条件下，构建III类塌陷口袋的亲水性氨基酸以减小口袋大小，这可能提高酶对小分子乙醇的亲和力，增加血液水平的乙醇酶活性。此外，我们在基因水平上研究了III类ADH的翻译调节。我们克隆了小鼠第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‘非编码区和10kb的3’非编码区，并完成了该基因全长的DNA序列分析。在约2kb的5‘非编码区发现了XRE、STAT和SRY等罕见序列作为转录因子的基序序列。这表明小鼠Class III ADH的表达受有毒化合物、激素和性别决定因素的调节，因此Class III ADH的活性受底物结合袋中因溶液疏水性改变而引起的结构变化和基因转录调控的调节，从而促进体内的酒精代谢和其他生理代谢。III型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。较少

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