Molecular cloning, baculovirus expression, and tissue distribution of the zebrafish aldehyde dehydrogenase 2.
Ethanol is metabolized to acetaldehyde mainly by the alcohol dehydrogenase pathway and, to a lesser extent, through microsomal oxidation (CYP2E1) and the catalase-H(2)O(2) system. Acetaldehyde, which is responsible for some of the deleterious effects of ethanol, is further oxidized to acetic acid by aldehyde dehydrogenases (ALDHs), of which mitochondrial ALDH2 is the most efficient. The aim of this study was to evaluate zebrafish (Danio rerio) as a model for ethanol metabolism by cloning, expressing, and characterizing the zebrafish ALDH2. The zebrafish ALDH2 cDNA was cloned and found to be 1892 bp in length and encoding a protein of 516 amino acids (M(r) = 56,562), approximately 75% identical to mammalian ALDH2 proteins. Recombinant zebrafish ALDH2 protein was expressed using the baculovirus expression system and purified to homogeneity by affinity chromatography. We found that zebrafish ALDH2 is catalytically active and efficiently oxidizes acetaldehyde (K(m) = 11.5 microM) and propionaldehyde (K(m) = 6.1 microM). Similar kinetic properties were observed with the recombinant human ALDH2 protein, which was expressed and purified using comparable experimental conditions. Western blot analysis revealed that ALDH2 is highly expressed in the heart, skeletal muscle, and brain with moderate expression in liver, eye, and swim bladder of the zebrafish. These results are the first reported on the cloning, expression, and characterization of a zebrafish ALDH, and indicate that zebrafish is a suitable model for studying ethanol metabolism and, therefore, toxicity.
Lassen
Natalie
N
Molecular Toxicology & Environmental Health Sciences Program, Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, 80262, USA.
Estey
Tia
T
Tanguay
Robert L
RL
Pappa
Aglaia
A
Reimers
Mark J
MJ
Vasiliou
Vasilis
V
D000444:Aldehyde Dehydrogenase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D015153:Blotting, Western; D001923:Brain Chemistry; D002492:Central Nervous System Depressants / Q000378:metabolism / Q000633:toxicity; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D014018:Tissue Distribution; D015027:Zebrafish / Q000378:metabolism*
D002492:Central Nervous System Depressants; D018076:DNA, Complementary; D000431:Ethanol; D000444:Aldehyde Dehydrogenase