Figure 2.

Rio1 has ATPase activity in vitro. (a) Hydroxylamine sensitivity of acyl-phosphate analysis. Phosphorimage of filter blot of ³²P-labeled ctRio1 after treatment with assay buffer, 0.5 M NaCl or 0.5 M hydroxylamine (b–d) ctRio1 ATPase (% of γ-phosphate release) and kinase (autophosphorylation) activities were tested in single turnover experiments using 1 μM of the indicated purified recombinant protein (load is shown in (d), lower panel). (b–c) Time-dependent free phosphate released (%Pi) as obtained from thin layer chromatography experiments of single turnover assays. (d) Comparative analysis of time-dependent autophosphorylation activity as obtained by SDS-PAGE and detected by autoradiography from single turnover experiments performed with 1 μM of the indicated purified recombinant protein. Note that the amount of sample loaded in (d) is 100 times more than in (b).