Fig. 7. RanGDP removes higher amounts of importin-ß from extracts of Ketel^D eggs than from extracts of wild-type (WT) eggs. RanQ69L protein binds higher amounts of importin-ß protein from WT egg extract than from extract of Ketel^D eggs. GST protein was used as a negative control (A, left). RanQ69L protein removes high amounts of purified WT importin-ß but not purified P446L mutant protein. At the same time RanGDP removes higher amounts of purified P446L importin-ß compared with the purified WT importin-ß (A, right). More Ran is precipitated with the anti-Ketel antibody from extracts of the Ketel^D eggs than from extracts of WT eggs. (B, left). However, if an energy-regenerating system and 3 µM (10 times the endogenous importin-ß concentration in the extract) purified wild-type or P446L mutant importin-ß are added to WT egg extract, more Ran is precipitated from the extract supplemented with WT importin-ß (B, right). Wild-type importin-ß inhibits both exchange of the labeled GTP from Ran (C) and GTP hydrolysis (D), whereas P446L mutant importin-ß has no effect on both nucleotide exchange and GTP hydrolysis. In C, the time course of nucleotide exchange is shown on a semi-logarithmic scale and D shows the results of the reactions performed in duplicate.

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