TABLE 2

Effects of taxoid site and laulimalide site drugs on tubulin assembly at 0°C

The 0.1-ml reaction mixtures contained one or two drugs, as indicated, 10 μM tubulin, 0.1 M MES, and 4% dimethyl sulfoxide. Incubation was for 90 min at 0°C, followed by centrifugation for 10 min at 30,000 rpm at 2°C (lowest temperature setting on the centrifuge). Incubation was on ice, and all mixing operations were performed in a 4°C cold room. Protein content of 20 μl of each supernatant and of an uncentrifuged reaction mixture was determined. All data were corrected for the amount of protein removed by centrifugation of reaction mixtures containing 4% dimethyl sulfoxide but no drug. The amount of protein removed by centrifugation without drug averaged 5.7 ± 2% in the experiments used to generate this table. Each experimental data point represents the average ± S.D. from three independent assays, except that only two data points were obtained for each assay condition containing laulimalide. The values shown without brackets when a single drug was present in the assay mixture were obtained with the drug at 20 μM. When drug mixtures were used, each drug was present at 10 μM. The values shown with brackets when a single drug was present in the assay mixture were obtained with the drug at 10 μM.