ABSTRACT
Rat liver contains two class 1 aldehyde dehydrogenases (ALDHs): a constitutive isozyme (ALDH1) and a phenobarbital-inducible isozyme (ALDH-PB). Defining characteristics of mammalian class 1 ALDHs include a homotetrameric structure, high expression in liver, sensitivity to the inhibitor disulfiram, and high activity for the oxidation of retinal. It is often presumed that ALDH-PB is the rat ortholog of mammalian ALDH1, and the identity of rat ALDH-PB is commonly interchanged with ALDH1. In this study, we characterized recombinant rat liver cytosolic ALDH1 and ALDH-PB. Previous reports indicate that ALDH-PB is a homodimer; however, we found by mass spectrometry and gel electrophoresis that it is a homotetramer. ALDH1 mRNA was highly expressed in untreated rat liver, while ALDH-PB had very weak expression, in contrast to a previous report that ALDH-PB mRNA is expressed in untreated rat liver. Rat liver ALDH1 had a high affinity for retinal (K(m) = 0.6 microM), while no oxidation by ALDH-PB could be detected with 20 microM retinal. ALDH1 was more efficient at oxidizing acetaldehyde, propionaldehyde, and benzaldehyde and was more sensitive to disulfiram inhibition. We conclude that rat liver ALDH1 is the ortholog of mammalian liver ALDH1. Furthermore, despite a high level of sequence identity and classification as a class 1 ALDH, ALDH-PB does not function like ALDH1. ALDH-PB is not merely an inducible ALDH1 isozyme; it is a distinct ALDH isozyme.
Subject(s)
Aldehyde Dehydrogenase/metabolism , Cytosol/enzymology , Isoenzymes/metabolism , Liver/enzymology , Phenobarbital/pharmacology , Sequence Homology, Amino Acid , Aldehyde Dehydrogenase/antagonists & inhibitors , Aldehyde Dehydrogenase/biosynthesis , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase 1 Family , Amino Acid Sequence , Animals , Cytosol/drug effects , Disulfiram/pharmacology , Enzyme Induction/drug effects , Enzyme Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/biosynthesis , Isoenzymes/genetics , Magnesium Chloride/pharmacology , Mass Spectrometry , Molecular Sequence Data , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Retinal Dehydrogenase , Substrate Specificity/drug effectsABSTRACT
The presence of a constitutively expressed aldehyde dehydrogenase (ALDH) in the rat liver cytosol is controversial (Tottmar et al., 1973; Lindahl and Evces, 1984; Berger and Weiner, 1977; Tank et al., 1981; Truesdale-Mahoney et al., 1981; Cao et al., 1989). A cDNA encoding a constitutively expressed rat liver cytosolic class 1 ALDH was cloned using a PCR-based strategy. The open reading frame consisted of 1503 nucleotides which encoded a protein of 501 amino acids. In order to compare the rat and human nucleotide sequences, we sequenced the entire open reading frame of a human liver cytosolic ALDH cDNA clone (Zheng et al., 1993). Rat liver constitutively expressed cytosolic ALDH was 99.7, 91.8, 89.0, and 83.8% identical to rat kidney, mouse liver, rat liver phenobarbital-inducible, and human liver cytosolic class 1 ALDH cDNAs, respectively. Northern blot analysis indicated that constitutively expressed rat cytosolic ALDH mRNA is expressed in lung, kidney, liver, skeletal muscle, and testis, with weak expression in heart and brain. These results strongly suggest that a constitutively expressed ALDH is present in rat liver cytosol.
