Nucleotide-binding rates and GTPase activities of Rab27a and the various mutants.

Nucleotide-binding and GTPase activity rates were measured by the filtration method, and the data are expressed as the percentage of the maximum radioactive nucleotide binding to Rab27a WT or mutant proteins. (A) GTP binding. The Rab27a (WT) and mutant (Thr23Asn, Trp73Gly, Leu130Pro, and Ala152Pro) proteins were incubated with (³H)-GTP for the indicated times, and the amount of bound (³H)-GTP was determined. (B) GDP binding. Fixation of (³H)-GDP with the same recombinant proteins. (C) GTPase activities of proteins WT (▪), Trp73Gly (gray triangle), and Gln78Leu (inverted gray triangle). The WT and mutant Rab27a proteins were loaded with (γ-³² P)GTP, and GDP hydrolysis was determined by the release of (³²P)Pi (phosphate inorganic). (D) GTP binding. Fixation of (³H)-GTP with 2 new mutations at position 73: Trp73Ser, Trp73Leu as performed in panel A. (E) GTPase activities of proteins Trp73Ser (gray square) and Trp73Leu (inverted gray triangle) performed in the same conditions as in panel C. The results show 3 different experiences performed in duplicates and analyzed by nonlinear regression with the program Prism 3 (Graph Pad Software, San Diego, CA).