The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates.
ATP, ubiquitin-dependent proteolysis proceeds through covalent intermediates between target proteins destined for degradation and the 8,600-Da polypeptide ubiquitin. The ubiquitin moiety therefore represents a sensitive immunological marker for the specificity and function of this novel post-translational modification. Methods are described for the immunochemical detection of ubiquitin conjugates immobilized on nitrocellulose filters following electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. A further modification allows quantitation of conjugated ubiquitin to the exclusion of free polypeptide. Comparisons of conjugate pools in rabbit reticulocytes and erythrocytes demonstrate that 83 +/- 3% and 31 +/- 0.2%, respectively, of total intracellular ubiquitin exists covalently bound to target proteins. Similar large proportions of conjugated ubiquitin were found in three tissue culture cell lines. Subcellular fractionation revealed that 25% of total ubiquitin conjugates of reticulocytes sediment with the 22,000 X g stromal fraction with the remainder found in the 100,000 X g supernatant. In contrast, significant levels of erythrocyte ubiquitin conjugates occur only in the 100,000 X g supernatant, suggesting ubiquitin-mediated proteolysis actively degrades stromal components lost during terminal maturation. Reticulocytes retain their full complement of active ubiquitin during maturation indicating the concomitant decline in energy-dependent proteolysis does not result from ubiquitin inactivation. That the lower level of ubiquitin conjugates and the accompanying rate of energy-dependent proteolysis in erythrocytes is a consequence of limited substrate availability is suggested by observed increases in conjugate pools and induction of specific ubiquitin-protein adducts on incubation with either phenylhydrazine or sodium nitrite.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001798:Blood Proteins / Q000032:analysis / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002522:Chlorocebus aethiops; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000097:blood*; D006651:Histocytochemistry; D007158:Immunologic Techniques; D008970:Molecular Weight; D010659:Phenylhydrazines / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012977:Sodium Nitrite / Q000494:pharmacology; D013347:Subcellular Fractions / Q000032:analysis; D014452:Ubiquitins / Q000097:blood*
D001798:Blood Proteins; D006609:High Mobility Group Proteins; D010659:Phenylhydrazines; D014452:Ubiquitins; C030299:phenylhydrazine; D000255:Adenosine Triphosphate; D003101:Collodion; D012977:Sodium Nitrite