Ubiquitin, which can conjugate with cellular proteins, is classified into two forms: free ubiquitin and multiubiquitin chains. The latter is active as a signal for degradation of the targeted proteins. We found both forms in human serum and, using two immunoassays, quantitated them in sera from healthy subjects and patients with some diseases. Because of putative leakage of erythrocyte ubiquitin, hemolytic serum and serum obtained after long incubation (>1–2 h) of blood at room temperature were excluded. Serum concentrations of multiubiquitin chains and free ubiquitin were substantially higher in rheumatoid arthritis and hemodialysis patients, respectively, than healthy subjects. Additionally, in acute viral hepatitis, serum multiubiquitin chain concentrations were increased in the acute phase, decreased in the recovery phase, and correlated with alanine and aspartate aminotransferase activities (r = 0.676 and 0.610, P <0.0001 and <0.001, respectively). Therefore, serum ubiquitin may have prognostic value.

A sandwich ELISA has been developed to measure intracellular levels of multi-ubiquitin chains. The mixture of multi-ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus "'I-ubiquitin) and lysozyme with ubiquitin-ligating enzymes and ATP, was partially purified and established as a standard named the multi-ubiquitin-chain reference preparation 1 (MUCRPI). The concentration of MUCRPI was calculated from the recovered radioactivity of "'I-ubiquitin. All measurements by the ELISA were expressed in terms of MUCRPI. The ELISA showed good sensitivity (98 pg/ml), precision (intra-assays < 6%) and reproducibility (interassay < 9%). In addition, there was no substantial cross-reaction with mono-, di-and tri-ubiquitin, or mono-ubiquitinated and di-ubiquitinated lysozyme in the ELISA, and large multi-ubiquitin chains ( n > approximately 6 ) may be fully reactive. These results combined with excellent results in the recovery and dilution tests guarantee accurate measurement of multi-ubiquitin chains in cell extracts prepared with a lysis buffer (water soluble) or the buffer supplemented 8 M urea (urea soluble). The level of the water-soluble multi-ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea-soluble chain level was higher in the reticulocytes. Heat-shock treatment of HeLa cells increased the urea-soluble multi-ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi-ubiquitin-conjugate turnover.Keywords: ELISA ; heat shock ; multi-ubiquitin chain; reticulocyte; ubiquitin.Ubiquitin is found in either a free form or a form bound covalently to intracellular proteins in vivo (Hershko, 1988;Finley and Chau, 1991). In the latter form, the ubiquitin C-terminus is ligated to &-amino groups of appropriate Lys residues in target proteins by a family of ubiquitin-conjugating enzymes: a ubiquitin-activating enzyme (El), a ubiquitin-carrier protein (E2), and a ubiquitin-protein ligase (E3) (Hershko et al., 1983;Hershko and Ciechanover, 1992). In this process, several ubiquitin moieties are usually ligated sequentially to the initial acceptor protein, forming multi-ubiquitin chains in which the ubiquitin Cterminus is conjugated to the &-amino group of Lys48 of another ubiquitin moiety (Chau et al., 1989). The multi-ubiquitin chain acts as a signal to induce degradation of target proteins by 2 6 s proteasome (Chau et al., 1989;Hershko and Ciechanover, 1992). Therefore, the chain structure is essential for ubiquitin-dependent proteolysis.The ubiquitin-dependent proteolysis has been implicated in a variety of cellular processes, including stress response (Finley et al., 1987;Carlson et a]., 1987), cell-cycle control (Glotzer et al., 1991;Nishizawa et al., 1993) and apoptosis (Delic et al., 1993). Intracellular levels of ubiquitin and ubiquitin-protein conjugates are changed in response to heat shock (Parag et al., Abbreviations. MUCRPI , multi-ubiquitin chain reference preparation 1 ; E l , ubiquitin-activating enzy...

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