Identification of the 37-kd rat liver protein that forms an acetaldehyde adduct in vivo as Δ 4-3-ketosteroid 5β-reductase



Acetaldehyde, the first product of alcohol metabolism, is highly reactive. Several proteins have been shown to be covalently modified by acetaldehyde in vivo. We have previously reported the detection of a cytosolic 37-kd protein-acetaldehyde adduct (-AA) in the liver of alcohol-fed rats. The liver extract from an alcohol-fed rat was subjected to 2-dimensional (2D) sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride (PVDF) membrane, and the 37-kd protein-AA spot was digested with trypsin and sequenced for amino acids. Degenerate oligonucleotides corresponding to a peptide sequence of the protein-AA were used as the probe to screen a λ gt11 rat liver complementary DNA (cDNA) library. A clone that extended to a potential ATG start codon was identified. The open reading frame was 978 nucleotides long, encoding 326 amino acid residues. The sequence matched that of rat liver Δ 4-3-ketosteroid 5β-reductase. The cloned cDNA was expressed in Escherichia coli using pGEX-KG as the vector. The expressed protein was found to be of correct molecular weight. It reacted with an antibody that recognized the unmodified liver 37-kd protein by Western blotting. Peptide profiles of tryptic- digested recombinant protein and the purified rat liver 37-kd protein were similar and yielded the same peptide sequence. Δ 4-3-ketosteroid 5β-reductase catalyzes the reduction of key intermediates during bile acid biosynthesis. Whether modification of the 5β-reductase by acetaldehyde affects the enzyme activity and bile acid synthesis remains to be studied.