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Kinetic Characterization of Two Classes of Dog Liver Alcohol Dehydrogenase Isoenzymes
Author(s) -
Bosron William F.,
Gaither James W.,
Magnes Leslie J.
Publication year - 1985
Publication title -
alcoholism: clinical and experimental research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.267
H-Index - 153
eISSN - 1530-0277
pISSN - 0145-6008
DOI - 10.1111/j.1530-0277.1985.tb05740.x
Subject(s) - alcohol dehydrogenase , isozyme , biochemistry , chemistry , alcohol , enzyme , biology
In order to relate the catalytic properties of alcohol dehydrogenase (ADH), the rate‐limiting enzyme for alcohol metabolism, with the pharmacokinetics of ethanol elimination in vivo, the multiple molecular forms of dog Hver ADH were purified and their steady state kinetics investigated. Two different classes of ADH forms were identified by starch gel electrophoresis: the class I isoenzymes migrate to the cathode and the class II forms migrate to the anode. Three different patterns of the cathodic class I isoenzymes were identified in different liver specimens. Three molecular forms were observed for patterns A and C, and five for B. The two classes of isoenzymes were separated by affinity chromatography and purified by column chromatography. The three predominant class I isoenzymes, A1, B2, and C1, in type A, B, and C livers, respectively, were isolated by high performance cation‐exchange chromatography. The steady state kinetic constants of the A1, B2, and C1 isoenzymes are similar, but differ substantially from those of the class II enzyme. The class II enzyme is much less sensitive to pyrazole inhibition, K i = 2 nw, than the class I forms, K i = 0.6 μM. Methanol is not a substrate for the class II enzyme, whereas it is oxidized by the class I isoenzymes. The class I isoenzymes exhibit a lower K., and substrate inhibition K, for ethanol, 0.4 and 160 nw, respectively, than values for the class II enzyme, 10 and 610 mi, respectively. The properties of class I and II dog liver ADH are similar to those of the respective isoenzymes purified from human and monkey liver. Based on kinetic data for the two classes of dog liver alcohol dehydrogenase, we suggest that the pharmacokinetics of ethanol elimination should obey a two term Michaeiis‐Menten model with substrate inhibition at high alcohol concentration.