Kinetic characterization of two classes of dog liver alcohol dehydrogenase isoenzymes.
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 liver 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, Ki = 2 mM, than the class I forms, Ki = 0.6 microM. 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 Km and substrate inhibition Ki for ethanol, 0.4 and 160 mM, respectively, than values for the class II enzyme, 10 and 610 mM, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D004285:Dogs; D000431:Ethanol / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008253:Macaca mulatta; D051379:Mice; D012453:Saimiri; D013045:Species Specificity
D007527:Isoenzymes; D000431:Ethanol; D000429:Alcohol Oxidoreductases