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Table I.

Kinetic Parameters of Drosophila Ral

Kd
GDP dissociation constant K−1 × 1,000 (min−1)
GDS sensitivity
GDPGTPγS−GDS+GDS
 nM   -fold 
Wild-type 14.0 30.7 8.6 39.6 4.6 
RalG20V 8.4 38.0 5.8 17.9 3.1 
RalS25N 95.1 406.0 86.6 86.6 1.0 
GTPase (steady-state rate) Kss × 1,000 (min−1 GTPase (actual catalytic rate) Kcat × 1,000 (min−1)
 
GAP sensitivity 
−GAP +GAP 

 
     -fold 
Wild-type 7.1  23.1 187.3 8.1 
RalG20V 3.4  21.1 22.7 1.1 
RalS25N 4.3  ND ND ND 
Kd
GDP dissociation constant K−1 × 1,000 (min−1)
GDS sensitivity
GDPGTPγS−GDS+GDS
 nM   -fold 
Wild-type 14.0 30.7 8.6 39.6 4.6 
RalG20V 8.4 38.0 5.8 17.9 3.1 
RalS25N 95.1 406.0 86.6 86.6 1.0 
GTPase (steady-state rate) Kss × 1,000 (min−1 GTPase (actual catalytic rate) Kcat × 1,000 (min−1)
 
GAP sensitivity 
−GAP +GAP 

 
     -fold 
Wild-type 7.1  23.1 187.3 8.1 
RalG20V 3.4  21.1 22.7 1.1 
RalS25N 4.3  ND ND ND 

To determine the Kd values of DRal mutants for the guanine nucleotides, DRal mutants (1 pmol) were incubated for various periods of time at 30°C with various concentrations of [−3H]GDP or [35S]GTPγS in 100 μl of reaction mixture (50 mM Tris/HCl, pH 7.5, 5 mM MgCl2, 1 mM DTT, and 1 mg/ml BSA; Kikuchi et al., 1988). To determine the RalGDS activity for DRal mutants, the [−3H]GDP-bound form of DRal mutants (8 pmol) were incubated with or without 200 nM RalGDS for various periods of time at 30°C. K−1 was determined as described (Shoji et al., 1989). Kss of the GTPase activity was determined by incubating DRal mutants with γ[32P]GTP for various periods of time at 30°C and expressed as the turnover number (Kikuchi et al., 1988). Kcat of GTPase activity was determined in the presence or absence of RalGAP (7 μg of protein) as described by Higashijima et al. (1987). Kss for DRalS25N was assayed at 5 μM GTP instead of 1 μM GTP, which was employed for the assay of wild-type DRal and DRalG20V. The Kcat of DRalS25N was not determined because most of the γ[32P]GTP was dissociated during these assays. The results shown are the means of three independent experiments.

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