Int. J. Environ. Res. Public Health 2010, 7 1079 assay using 15 mM ethanol as a substrate [33]. However, when the ADH3 fraction was separated from other ADH isozymes of mouse liver by CM-chromatography, it was found to account for about 13% of total liver ADH activity; ADH1 accounts for more than 62%, and ADH2 accounts for only 2%. Moreover, as the concentration of substrate ethanol increased, the percentage of the ADH3 fraction increased while that of the ADH1 fraction decreased. So, we speculated that ADH3 plays a significant role in alcohol metabolism [24]. 4. Activation of ADH3 by Solution Hydrophobicity and Dynamic Metabolism of Alcohol Mouse ADH1 shows the largest activity towards ethanol around 10 mM (Km ≒ 1 mM), and the activity decreases in a dose-dependent manner as the concentration becomes higher. In contrast, ADH3 shows a low activity towards ethanol at several tens of millimoles, but the activity increases linearly up to the molar level (Km < 1 M) [15,16]. However, we have sometimes observed that ADH3 is activated during the purification process when measured by the standard ADH assay. By investigating the regulation of its activity under various conditions, we found that solution hydrophobicity markedly activated ADH3 and increased its catalytic efficiency (kcat/Km) for ethanol [20,25]. Tert-butanol (C4), which strongly induces solution hydrophobicity in a medium due to its strong hydrophobic hydration and its high miscibility, increases the kcat/Km of ADH3 almost ten-fold, lowering its Km for ethanol. The Km decreases below 100 mM by solution hydrophobicity induced with tert-butanol above 1.8 M (unpublished data). Since we observed no significant changes in the secondary, tertiary, or quaternary structures of mouse ADH3 in hydrophobic solution, we concluded that solution hydrophobicity induces a slight structural change in the substrate-binding pocket of ADH3 [25]. Indeed, the reason why ADH3 has a low activity for ethanol is that the volume of its substrate-binding pocket is larger than that of ADH1 due to the substitution of smaller, more hydrophilic amino acid residues [35]. The hydrophilic amino acids constituting the pocket of ADH3 collapse in a hydrophobic medium, thus, reducing the size of the pocket. This structural change may raise its affinity for a small molecule like ethanol, thereby increasing its ability to oxidize ethanol. Thus, solution hydrophobicity lowers the Km of ADH3 and increases it activity with respect to ethanol. The increase in activity in a hydrophobic solution is specific to ADH3; all other ADH isozymes show a decrease in activity in this type of medium [20]. In contrast to water molecules in vitro, intracellular ones are dynamically restrained by their hydrophobic interaction with various macromolecules and intracellular membranes with an extensive reticulum [36]. When Nile red, a fluorescent probe for hydrophobicity, is added to the cytoplasm of mouse liver, its spectrum is red-shifted from the spectrum obtained when it is added to a lipid droplet or a membrane of the liver, reflecting the hydrophobicity of a cytoplasmic solution [25]. Thus, also in vivo, ADH3 should be activated and ADH1 activity should be depressed by solution hydrophobicity in the liver cells. It is reasonable to conclude that both ADHs have dynamic roles in alcohol metabolism, with their activities being inversely regulated by changes in the solution hydrophobicity of liver cells.