Aldehyde Dehydrogenases: Measurement of Activities and Protein Levels
St. Cloud State University, Saint Cloud, Minnesota.Current protocols in toxicology 12/2005; Chapter 4:Unit4.18. DOI: 10.1002/0471140856.tx0418s26
Seventeen enzymes are currently viewed as belonging to the human aldehyde dehydrogenase superfamily, and all of them catalyze the pyridine nucleotide-dependent oxidation of aldehydes to acids. Depending on the specific aldehyde dehydrogenase, lack of sufficient catalytic activity (1) results in a gross pathological phenotype in the absence of any insult, or (2) is ordinarily of no consequence with respect to gross phenotype, but is of consequence when the organism is subjected to a relevant insult. Described in this unit are eight assays that can be used to (semi)quantify various in vitro aldehyde dehydrogenase protein and/or catalytic activity levels, and two that can be used to semiquantify various in situ aldehyde dehydrogenase protein and/or catalytic activity levels. Aldehyde dehydrogenases also catalyze the hydrolysis of esters; this unit includes an assay that can be used to quantify that catalytic activity as well. Preparation of test materials and of antibodies to the aldehyde dehydrogenases are described in three support protocols.
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ABSTRACT: Ethylene glycol ethers (EGEs) are primary alcohols commonly used as solvents in numerous household and industrial products. Exposure to EGEs has been correlated with delayed encephalopathy, metabolic acidosis, sub-fertility and spermatotoxicity in humans. In addition, they also cause teratogenesis, carcinogenesis, hemolysis, etc., in various animal models. Metabolism EGEs parallels ethanol metabolism, i.e., EGEs are first converted to 2-alkoxy acetaldehydes (EGE aldehydes) by alcohol dehydrogenases, and then to alkoxyacetic acids by aldehyde dehydrogenases (ALDHs). The acid metabolite of EGEs is considered responsible for toxicities associated with EGEs. The role of human ALDHs in EGE metabolism is not clear; accordingly, we have investigated the ability of five different human ALDHs (ALDH1A1, ALDH2, ALDH3A1, ALDH5A1 and ALDH9A1) to catalyze the oxidation of various EGE aldehydes. The EGE aldehydes used in this study were synthesized via Swern oxidation. All of the human ALDHs were purified from human cDNA clones over-expressing these enzymes in E. coli. The ALDHs tested, so far, differentially catalyze the oxidation of EGE aldehydes to their corresponding acids (K(m) values range from approximately 10 microM to approximately 20.0mM). As judged by V(max)/K(m) ratios, short-chain alkyl-group containing EGE aldehydes are oxidized to their acids more efficiently by ALDH2, whereas aryl- and long-chain alkyl-group containing EGE aldehydes are oxidized to their acid more efficiently by ALDH3A1. Given the product of ALDH-catalyzed reaction is toxic, this process should be considered as a bio-activation (toxification) process.
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