creased catabolism of ubiquitin and its conjugates. Our Hepatic protein accumulation during ethanol adminfindings also provide no indication that the ethanol-elicistration may result partly from an ethanol-elicited deited reduction in hepatic proteolysis is because of a ubicline in hepatic protein degradation, which we have prequitin-mediated mechanism. (HEPATOLOGY 1996;23:1556viously shown. We conducted the current studies to 1563.) 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 catabo-Hepatomegaly is common in alcoholics and alcohollism. Rats were pair-fed liquid diets containing either fed experimental animals. It is closely associated with ethanol (36% of calories) or isocaloric maltose-dextrin increased levels of heptocellular protein that contribute for 1 to 5 weeks. Ubiquitin was immunochemically quanto hepatocyte hypertrophy. [1][2][3][4] Investigations into the tified by competitive enzyme-linked immunosorbent reasons for ethanol-induced net protein gain have proassay (ELISA) in crude cytosol fractions from whole liver duced variable results. Though some studies have reand in 12,000g supernatants of hepatocyte lysates. Ubiported that ethanol consumption increases protein synquitin levels in hepatic cytosol fractions of ethanol-fed thesis, 4,5 other reports indicate that hepatic protein rats exceeded those of controls by about 30%. Isolated hepatocytes from ethanol-fed animals also showed a 40% synthetic activity is unaffected or decreased by acute to 75% elevation of ubiquitin above that in cells of pairor chronic ethanol administration. 2,3,6,7 Protein catabofed controls and this difference exceeded the relative lism, which has an equal, if not greater, role than synrise in hepatocellular protein. In hepatocyte lysates subthesis in regulating cellular protein levels, is decreased jected to sodium dodecyl sulfate-polyacrylamide gel by ethanol consumption. 8-10 The mechanism(s) responelectrophoresis (SDS-PAGE) and immunoblotting, we sible for this decline are unclear. Most intracellular detected monomeric ubiquitin and higher molecular proteins are degraded in lysosomes, 11 but an equally mass ubiquitin-protein conjugates. However, the immuimportant extralysosomal proteolytic pathway, with noblot analyses revealed no quantitative changes in the greater specificity, is mediated by ubiquitin.
level of either free or conjugated ubiquitin. The ubiqui-
Ubiquitin is found in all eukaryotic cells. It is a tin conjugating activity of crude and diethyl aminoethyl-fractionated liver cytosols of ethanol-fed rats had highly conserved 76-amino acid protein (relative molecequal capacities to those from controls in catalyzing the ular mass Γ
8.5 kd) that covalently binds proteins desformation of ubiquitin-protein conjugates. Our findings tined for degradation. Three enzymes, E1, E2, and E3, indicate that chronic ethanol consumption increased catalyze the formation of ubiquitin-protein conjugates, the level of immunoreactive ubiquitin in rat liver. This a process called ubiquitylation (for reviews, see refermay have resulted from enhanced ubiquitin production ences 12-14). Cellular proteins targeted for degradation because of an ethanol-elicited stress response and/or deare ubiquitylated at their NH 2 termini and at the eamino groups of lysine residues within the polypeptide chain. These conjugation points may each carry one Abbreviations: IgG, immunoglobulin G; ELISA, enzyme-linked immunosor-ubiquitin molecule or a multi-ubiquitin chain. 15,16 Probent assay; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoteins that are multi-ubiquitylated are more easily deresis; EDTA, ethylenediaminetetraacetic acid. graded than monoubiquitylated proteins by the 26S
From the Liver Study Unit, Omaha Veterans Affairs Medical Center and proteasome that catalyzes hydrolysis of the protein