The highly active
alcohol dehydrogenase (EC 1.1.1.1) in rat hepatic
tumor cells (HTC) was purified 120-fold by chromatography on
DEAE-
Sepharose and
AMP-Agarose to yield an
enzyme with a specific activity of 88 mumole/min/mg
protein, assayed with 1.7 mM NAD+ and 0.55 M
ethanol at pH 9 and 30 degrees C. (By comparison, purified, normal rat liver
enzyme has an activity of about 1 unit/mg.) Based on its physical and kinetic properties, we conclude that the HTC
isozyme is the same as the
enzyme from rat stomach and another rat
hepatoma (Cederbaum AI, Pietruszko R, Hempel J, Becker FF, and Rubin E (1975) Arch Biochem Biophys 171:348-360). The kinetics of the HTC
enzyme are consistent with the Ordered Bi Bi mechanism. The kinetic constants are generally much larger for the HTC
enzyme than for the normal rat liver
enzyme. The Michaelis constants for
ethanol and
acetaldehyde (Kb = 1100 mM, Kp = 260 mM) are 1000-fold larger, and the constants for
NADH are 10 to 50-fold larger. Although the HTC
enzyme has low catalytic efficiency (V/Kb) on
ethanol, it has much better activity on longer chain
alcohols, but no activity on
cyclohexanol. The pH dependence of V/Kb with
ethanol is unusual in that it appears to be a linear function of pH, increasing with a slope of 0.56. Thus, the active sites of the liver and HTC
enzymes may be different, although the HTC
enzyme is inactivated by
bromoacetate and bipyridine as is found for the liver
enzyme. The HTC (stomach)
enzyme may function to oxidize high concentrations of ingested
ethanol or longer chain
alcohols.