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

Abstract

After alcohol ingestion, ethanol is oxidized to acetalAcetaldehyde, the first product of alcohol metabolism, is highly reactive. Several proteins have been shown to dehyde in the liver mainly by the action of alcohol dehybe covalently modified by acetaldehyde in vivo. We have drogenase 1 and, on prolonged alcohol consumption, by previously reported the detection of a cytosolic 37-kd microsomal cytochrome P450IIE1. 2 Little acetaldehyde protein-acetaldehyde adduct (-AA) in the liver of alcohol-accumulates during alcohol oxidation because this infed rats. The liver extract from an alcohol-fed rat was termediate is rapidly converted to acetate by aldehyde subjected to 2-dimensional (2D) sodium dodecylsulfate dehydrogenase. Nevertheless, acetaldehyde is highly (SDS)-polyacrylamide gel electrophoresis (PAGE), trans-reactive. For many years, investigators have shown ferred to polyvinylidene difluoride (PVDF) membrane, that acetaldehyde can covalently modify proteins in and the 37-kd protein-AA spot was digested with trypsin vitro to form protein-acetaldehyde adducts (-AAs). 3 The and sequenced for amino acids. Degenerate oligonucleoformation of protein-AAs in vivo has been demontides corresponding to a peptide sequence of the proteinstrated more recently. 4-9 Our laboratory was the first AA were used as the probe to screen a lgt11 rat liver to report the detection of a liver protein-AA (apparent complementary DNA (cDNA) library. A clone that extended to a potential ATG start codon was identified. MW, 37,000) by Western immunoblot in rats fed alcohol The open reading frame was 978 nucleotides long, encod-chronically. 4 The 37-kd liver protein-AA can be deing 326 amino acid residues. The sequence matched that tected in rats within 1 week of alcohol feeding (either of rat liver D 4 -3-ketosteroid 5b-reductase. The cloned the high-fat Lieber-DeCarli or the low-fat AIN'76 liquid cDNA was expressed in Escherichia coli using pGEX-KG diet containing alcohol [Bio-Serv, Inc., Frenchtown, as the vector. The expressed protein was found to be of NJ]). 10 The bond between the 37-kd protein and acetalcorrect molecular weight. It reacted with an antibody dehyde was very stable, and its stability was not enthat recognized the unmodified liver 37-kd protein by hanced by incubating with cyanoborohydride as a reWestern blotting. Peptide profiles of tryptic-digested re-ducing agent. 4 Adding pyrazole (an inhibitor of alcohol combinant protein and the purified rat liver 37-kd pro-dehydrogenase) 11,12 to the alcohol-containing diet abro- dehydrogenase) 14 raised plasma acetaldehyde concentrations more than sixfold and greatly increased the intensity of the 37-kd protein-AA band on immunoblots. 13 These experiments therefore provide strong Abbreviations: AA, acetaldehyde adduct; KLH, keyhole limpet hemocyanin; evidence that the 37-kd band detected by immunoblot-SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; 2D, ting is truly a protein adduct of acetaldehyde. On subtwo-dimensional; Ig, immunoglobulin; PVDF, polyvinylid...

Acetaldehyde-modified and 4-hydroxynonenal-modified proteins in the livers of rats with alcoholic liver disease

“…37-kd protein is a constitutive part of the immunogen. 13 The immuIn contrast, the presence of 4-HNE adducts does not show noglobulin G fraction of the anti-PAA and the anti-37-kd liver protein antibodies were isolated using a Protein-A affinity column association with liver injury. (Pharmacia Fine Chemicals, Piscataway, NJ).…”

“…PAA and HPA were sured by headspace-gas chromatography method using a gas-chromatograph column (Poropak Q; Supelco, Inc., Bellefonte, PA) detected by Western blot analysis following the same standard procedures used previously in our laboratory. 13 Briefly, liver extracts equipped with a flame-ionization detector. Detailed procedures have been described previously.…”

“…26 Thus, the use of these different dietary body was raised in a rabbit using the 37-kd PAA purified from the models allowed us to study the relationship between adduct liver of an alcohol-fed rat by two-dimensional gel electrophoresis. 13 formation and the severity of liver injury. Results from this The anti-37-kd PAA antibody recognizes the unmodified 37-kd study indicate that the formation of the 37-kd PAA in the liver protein from control rats fed alcohol-free diets because the liver correlates with the severity of alcoholic liver injury.…”

Proteome analysis of rat hepatomas: Carcinogen-dependent tumor-associated protein variants

“…The high sequence identity of 3a-HSDs and 5b-Red suggests that these distinct activities arose through divergent evolution from a common ancestral protein [39]. Two major spots differed in one amino acid at position 190 (Thr?Ser) and two genes have been discussed [40]. Three different spots of 5b-Red have been described after 2-DE separations of rat liver tissue proteins.…”

Acetaldehyde-modified and 4-hydroxynonenal-modified proteins in the livers of rats with alcoholic liver disease

“…5,12 The 37-kd liver protein that forms acetaldeethanol, whereas rats fed MCTs and ethanol showed the hyde adduct in vivo has been identified recently as D 4 -3-weakest intensity. The 37-kd protein-adetaldehyde adduct ketosteroid-5b reductase, 13 a key enzyme involved in bile was located mainly in the pericentral region of the liver. No acid syntheses.…”