FIG. 4.

PA700 inactivates ODC in the presence of the 20S proteasome but not in the absence of the proteasome. Purified PA700 was separated by glycerol density gradient centrifugation. The gradient was separated into 30 fractions of 1 ml each. Samples of 150 μl of the gradient fractions were precipitated with 750 μl of acetone, and the precipitates were subjected to SDS-PAGE and stained with Coomassie blue (A). Twenty microliters of the samples were assayed for proteasome activity (B) and ODC degradation (C) and inactivation (D) in the presence (■) and absence (□) of purified 20S proteasome. For the ODC inactivation assay, samples were preincubated with clasto-lactacystin β-lactone (200 μM) at 37°C for 15 min. The inset in panel B shows SDS-PAGE of the 20S proteasome used.