Identification of the 37-kd rat liver protein that forms an acetaldehyde adduct in vivo as delta 4-3-ketosteroid 5 beta-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 lambda 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 delta 4-3-ketosteroid 5 beta-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. delta 4-3-ketosteroid 5 beta-reductase catalyzes the reduction of key intermediates during bile acid biosynthesis. Whether modification of the 5 beta-reductase by acetaldehyde affects the enzyme activity and bile acid synthesis remains to be studied.
Department of Medicine, Indiana University School of Medicine, IN., USA.
Y
Y
Zhu
M J
MJ
Fillenwarth
D
D
Crabb
L
L
Lumeng
R C
RC
Lin
D000079:Acetaldehyde / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D008565:Membrane Proteins; D010088:Oxidoreductases; C021936:3-oxo-5 beta-steroid delta 4-dehydrogenase; D000079:Acetaldehyde