Immunochemical analysis of the turnover of ubiquitin-protein conjugates in intact cells. Relationship to the breakdown of abnormal proteins.
Previous studies in a cell-free proteolytic system from reticulocytes indicated that the conjugation of ubiquitin with proteins plays a role in protein breakdown. To examine some of the physiological functions of the ubiquitin conjugation system, and immunochemical method was developed for the isolation of ubiquitin-protein conjugates from intact cells. A specific antiserum was raised against ubiquitin and purified by affinity chromatography on ubiquitin-Sepharose. When cells are labeled with tryptophan (which is missing from ubiquitin), labeled immunoreactive material isolated by the antibody is derived from the protein moiety of ubiquitin-protein conjugates. There is a marked increase in the labeling of ubiquitin-protein conjugates during the formation of abnormal proteins in reticulocytes (induced by the incorporation of amino acid analogs), suggesting that proteins with abnormal structure are more readily conjugated to ubiquitin than most normal proteins. Essentially similar, although less marked, effects of amino acid analogs were observed in Ehrlich ascites cells. When further protein synthesis was blocked with cycloheximide, ubiquitin conjugates decayed more extensively than the corresponding average labeled cellular proteins. This is consistent with the interpretation that a considerable part of ubiquitin conjugates is derived from a pool of rapidly degradable proteins.
D000818:Animals; D000936:Antigen-Antibody Complex; D001798:Blood Proteins / Q000378:metabolism; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D014452:Ubiquitins
D000936:Antigen-Antibody Complex; D001798:Blood Proteins; D002868:Chromosomal Proteins, Non-Histone; D007106:Immune Sera; D014452:Ubiquitins