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 extral ysosomal protein catabolism. Rats were pair-fed liquid diets containin g 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 f rom whole liver and in 12,000g supernatants of hepatocyte lysates. Ubi quitin levels in hepatic cytosol fractions of ethanol-fed rats exceede d those of controls by about 30%. Isolated hepatocytes from ethanol-fe d animals also showed a 40% to 75% elevation of ubiquitin above that i n cells of pair-fed controls and this difference exceeded the relative rise in hepatocellular protein. In hepatocyte lysates subjected to so dium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, we detected monomeric ubiquitin and higher molecular mass ubiquitin-protein conjugates. However, the immunoblot analyses re vealed no quantitative changes in the level of-either free or conjugat ed ubiquitin. The ubiquitin conjugating activity of crude and diethyl aminoethyl-fractionated liver cytosols of ethanol-fed rats had equal c apacities to those from controls in catalyzing the formation of ubiqui tin-protein conjugates. Our findings indicate that chronic ethanol con sumption 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 ubi quitin and its conjugates. Our findings also provide no indication tha t the ethanol-elicited reduction in hepatic proteolysis is because of a ubiquitin-mediated mechanism.