The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates

• Published in: The Journal of biological chemistry Vol. 260; no. 23; pp. 12464 - 12473
• Main Authors: Haas, A L, Bright, P M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15.10.1985; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphate - pharmacology; Animals; Biological and medical sciences; Blood Proteins - analysis; Blood Proteins - metabolism; Cell Fractionation; Cell Line; Cell metabolism, cell oxidation; Cell physiology; Chlorocebus aethiops; Collodion; Electrophoresis, Polyacrylamide Gel; Erythrocytes - metabolism; Fundamental and applied biological sciences. Psychology; High Mobility Group Proteins - blood; Histocytochemistry; Immunologic Techniques; Molecular and cellular biology; Molecular Weight; Phenylhydrazines - pharmacology; Protein Processing, Post-Translational; Rabbits; Reticulocytes - metabolism; Sodium Nitrite - pharmacology; Subcellular Fractions - analysis; Ubiquitins - blood; Assay; Proteins; Cell culture; Vertebrata; Mammalia; Animal; Identification; Immunochemistry; Complexes; Immunological method
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)38895-6

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.