Effects of ethanol administration on components of the ubiquitin proteolytic pathway in rat liver.
Hepatic protein accumulation during ethanol administration may result partly from an ethanol-elicited decline in hepatic protein degradation, which we have previously shown. We conducted the current studies to examine the effects of ethanol administration on the levels of hepatic ubiquitin, an 8.5-kd protein which is an important mediator of extralysosomal protein catabolism. Rats were pair-fed liquid diets containing either ethanol (36% of calories) or isocaloric maltose-dextrin for 1 to 5 weeks. Ubiquitin was immunochemically quantified by competitive enzyme-linked immunosorbent assay (ELISA) in crude cytosol fractions from whole liver and in 12,000g supernatants of hepatocyte lysates. Ubiquitin levels in hepatic cytosol fractions of ethanol-fed rats exceeded those of controls by about 30%. Isolated hepatocytes from ethanol-fed animals also showed a 40% to 75% elevation of ubiquitin above that in cells of pair-fed controls and this difference exceeded the relative rise in hepatocellular protein. In hepatocyte lysates subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, we detected monomeric ubiquitin and higher molecular mass ubiquitin-protein conjugates. However, the immunoblot analyses revealed no quantitative changes in the level of either free or conjugated ubiquitin. The ubiquitin conjugating activity of crude and diethyl aminoethyl-fractionated liver cytosols of ethanol-fed rats had equal capacities to those from controls in catalyzing the formation of ubiquitin-protein conjugates. Our findings indicate that chronic ethanol consumption increased the level of immunoreactive ubiquitin in rat liver. This may have resulted from enhanced ubiquitin production because of an ethanol-elicited stress response and/or decreased catabolism of ubiquitin and its conjugates. Our findings also provide no indication that the ethanol-elicited reduction in hepatic proteolysis is because of a ubiquitin-mediated mechanisms.
Born
L J
LJ
Liver Study Unit, Omaha Veterans Affairs Medical Center, NE, USA.
Kharbanda
K K
KK
McVicker
D L
DL
Zetterman
R K
RK
Donohue
T M
TM
D000818:Animals; D003600:Cytosol / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000431:Ethanol / Q000633:toxicity*; D015151:Immunoblotting; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D011506:Proteins / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D014452:Ubiquitins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D011506:Proteins; D014452:Ubiquitins; D000431:Ethanol