MATERIALS AND METHODS

Isolation and Purification of 26S and 20S Proteasomes. The 26S proteasomes were prepared from washed rabbit reticulocytes (Green Hectares Farms, Oregon, WI). The cells were lysed with 1.5 volumes of 1 mM dithiothreitol (DTT) 1 and gentle shaking at 4 C for 10 min. The crude lysate was made 1 mM in ATP and 5 mM in MgCU and clarified by centrifugation at lOOOOOg for 60 min. The supernatant was recentrifuged at lOOOOOg for 5 h. The pellet containing

1 Abbreviations: DTT= dithiothreitol; SDS= sodium dodecyl sulfate; PAGE= polyacrylamide gel electrophoresis; Ub= ubiquitin; Ubal= ubiquitin aldehyde; Ubi-a, Ub2-a, and Ub3, 4-a= ubiquitini-125I-a-globin, ubiquitin-125I-a-gIobin, and ubiquitin, 4-125I-a-globin, ie, the conjugates of one, two, and a mixture of three and four molecules of ubiquitin, respectively, with one molecule of 125I-labeled a-globin. In this work, the conjugates can only be detected because of their 125I-label, and hence, the theoretical M, given for each (see text) includes the weight of 1 mol of 125I.