Abstract
Abstract The aldehyde dehydrogenases from livers of the inbred mouse strains C57BL/6J, DBA/2J, and the F1 generation of the cross between these strains (C57BL/6J x DBA/2J) have been purified over 60-fold. Alcohol dehydrogenase activity is not present in the purified enzyme preparation. Enzyme preparations from both strains and from the F1 cross specifically require NAD but can oxidize a variety of aldehyde substrates. Results obtained previously, using a crude liver homogenate as enzyme source and indoleacetaldehyde as substrate, demonstrate a 2- to 3-fold greater aldehyde-oxidizing capacity in the liver extracts of the C57BL/6J strain compared to similar extracts from the DBA/2J mice. Mice of the former strain prefer a 10% solution of alcohol to water, whereas DBA/2J mice avoid alcohol in this preference test. F1 generation mice are intermediate to the parents with regard to alcohol preference and aldehyde-oxidizing capacity. The purified enzymes from the two inbred sources are indistinguishable as determined by disc gel electrophoresis, ion exchange chromatography, and gel filtration criteria. Nevertheless, the aldehyde dehydrogenases of C57BL/6J and DBA/2J mice exhibit 2- to 3-fold differences in the Michaelis constants of aldehyde substrates. Reaction velocity differences between the two enzyme preparations are particularly apparent when acetaldehyde is the substrate. The C57BL/6J aldehyde dehydrogenase is activated by 10-6 m acetaldehyde to a reaction velocity greater than 2 times its original value, whereas the DBA/2J aldehyde dehydrogenase exhibits substrate activation only above 10-3 m acetaldehyde. Finally, it has been demonstrated by chromatographic analysis that, following intraperitoneal ethanol injection, acetaldehyde accumulates in the blood of DBA/2J mice to a greater extent than it does in C57BL/6J mice. This observation lends support to the possibility that the lower aldehyde dehydrogenase activity of the DBA/2J mice results in an acetaldehyde-induced avoidance of ethanol.
Highlights
The enzyme activity retained by the DEAE-cellulose column was purified further
Neither of the two mouse liver fractions isolated by DEAE-cellulose chromatography is activated by lop5 M diethylstilbestrol, which effects a 150% increase of the dehydrogenase activity in the enzyme preparation from rabbit liver [30]
The small percentage (10 to 20%) of aldehyde dehydrogenase activity that voids the DEAE-cellulose anion exchange column corresponds in amount to that aldehyde dehydrogenase activity found to reside in the mitochondrial particulate fraction [16]
Summary
Acetaldehyde was glass distilled prior to use. Other aldehydes were reagent grade from Matheson, Coleman and Bell. NAD, analogues of NAD, the bisulfite salt of indoleacetaldehyde, and protamine sulfate were purchased from Sigma. Dithiothreitol was obtained from Calbiochem, enzyme grade ammonium sulfate from Mann, DE-52 from Whatman, Cellex-C and Agarose from Bio-Rad, polyacrylamide gel materials from Eastman, and Sephadex G-25, coarse, from Pharmacia. Double glass-distilled water was used for all solutions. C57BL/BJ and DBA/BJ mice were purchased from The Jackson Laboratory, Bar Harbor, Maine, at an age of 6 to 8 weeks. Mice needed for pilot experiments were obtained from the Institute for Behavioral Genetics, Boulder, Colorado. Livers were removed by excision after cervical dislocation and immediately put into ice. Purification steps were started at once
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