Serum Ubiquitin-Protein Conjugates in Normal Subjects and Patients With Alcoholic Liver Diseases: Immunoaffinity Isolation and Electrophoretic Mobility

Abstract

Background We previously reported that levels of multiubiquitin chains, representing ubiquitin-protein conjugates, were significantly higher in sera of patients with alcoholic liver cirrhosis than in normal subjects and patients with other types of alcoholic liver disease. Methods To characterize them, ubiquitin-immunoreactive proteins were purified from sera of healthy human volunteers and patients with alcoholic liver diseases by using affinity chromatography on a Sepharose column containing an immobilized monoclonal antibody recognizing conjugated ubiquitin. Results SDS-PAGE analysis followed by Western blotting revealed that the immunoaffinity-purified proteins mainly contained multiple components with molecular masses greater than 60 kDa, almost all of which were immunostained with the ubiquitin antibody. This size heterogeneity was in agreement with the property of ubiquitin-protein conjugates in all cells examined. These results indicate that the immunoaffinity-purified serum proteins are polyubiquitinated proteins presumably derived from some somatic cells. These ubiquitinated proteins obtained from the alcoholic cirrhosis patients were stained more strongly than those from the normal subjects and patients with other types of alcoholic liver disease, although equal amounts of multiubiquitin chains were analyzed simultaneously. In addition, marked differences were observed in the two-dimensional PAGE pattern of the ubiquitin-protein conjugates purified from the alcoholic cirrhosis patient serum compared with those from the normal human serum: four distinct broad spots (presumably polyubiquitin-protein complexes) were observed only in the former. Conclusions This is the first report on isolation of ubiquitin-protein conjugates from human serum, and it indicates that not only their levels, but also their molecular compositions, were greatly affected by alcoholic cirrhosis.