ABILITY OF UBIQUITIN RADIOIMMUNOASSAY TO DISCRIMINATE BETWEEN MONOUBIQUITIN AND MULTI-UBIQUITIN CHAINS

Free ubiquitin (mainly monoubiquitin) and multi-ubiquitin chains coexi st in eukaryote cells and serve distinct cellular roles. However, any immunoassay systems established previously have not been proved to be applicable for measuring the former without cross-reactive responses w ith the latter. For this purpose, we developed a radioimmunoassay spec ific to monoubiquitin by employing antiserum US-1 against ubiquitin. I n this assay, ubiquitin-protein conjugates, prepared by a reticulocyte lysate fraction II and fractionated on Moro Q and Superdex 200 column s, exhibited practically no cross-reactivity. The cross-reactivity of fractionated ubiquitin-lysozyme conjugates was also analyzed as a func tion of their multi-ubiquitin chain size. As a result, the larger the conjugates were found to be, the weaker were the cross-reactive respon ses they showed, and the multi-ubiquitin chains (n > approx. 20) were substantially unreactive in the radioimmunoassay. By using the radioim munoassay, heat-shock-induced decrease in the level of cellular free ( mono)ubiquitin was detected. in addition, the standard preparation of multi-ubiquitin chains was not cross-reactive in all other five radioi mmunoassays employing distinct antibodies to ubiquitin (four antisera and a monoclonal antibody). These data suggest that radioimmunoassays employing ubiquitin antibodies raised by the general methods can discr iminate between monoubiquitin and multi-ubiquitin chains and quantitat e cellular free ubiquitin.