Identification of the 37-kd rat liver protein that forms an acetaldehyde adduct in vivo as Δ4-3-ketosteroid 5β-reductase

Yongyi Zhu, Michael J. Fillenwarth, David Crabb, Lawrence Lumeng, Renee C. Lin

Research output: Contribution to journalArticle

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Abstract

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 λgt11 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 Δ4-3-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. Δ4-3-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.

Original languageEnglish
Pages (from-to)115-122
Number of pages8
JournalHepatology
Volume23
Issue number1
DOIs
StatePublished - 1996

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Acetaldehyde
Liver
Proteins
Alcohols
Bile Acids and Salts
Peptides
Complementary DNA
Amino Acids
Liver Extracts
Initiator Codon
3-ketosteroid reductase
Gene Library
Recombinant Proteins
Oligonucleotides
Trypsin
Open Reading Frames
Polyacrylamide Gel Electrophoresis
Oxidoreductases
Nucleotides
Clone Cells

ASJC Scopus subject areas

  • Hepatology

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Identification of the 37-kd rat liver protein that forms an acetaldehyde adduct in vivo as Δ4-3-ketosteroid 5β-reductase. / Zhu, Yongyi; Fillenwarth, Michael J.; Crabb, David; Lumeng, Lawrence; Lin, Renee C.

In: Hepatology, Vol. 23, No. 1, 1996, p. 115-122.

Research output: Contribution to journalArticle

Zhu, Yongyi ; Fillenwarth, Michael J. ; Crabb, David ; Lumeng, Lawrence ; Lin, Renee C. / Identification of the 37-kd rat liver protein that forms an acetaldehyde adduct in vivo as Δ4-3-ketosteroid 5β-reductase. In: Hepatology. 1996 ; Vol. 23, No. 1. pp. 115-122.
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abstract = "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 λgt11 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 Δ4-3-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. Δ4-3-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.",
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