GTPase activity assay

G-protein a-subunits also possess specific residues that can be covalently modified by bacterial toxins. Cholera toxin catalyzes the transfer of ADP-ribose moiety of NAD to a specific arginine residue in certain a-subunits, whereas pertussis toxin ADP-ribosylates those a-subunits that contain a specific cysteine residue near the carboxy-terminus. Modification of the a-subunit by cholera toxin persistently activates these protein by inhibiting their GTPase activity, whereas pertussis toxin inactives Gia protein and thereby results in the uncoupling of receptor from the effector. G-protein a-subunits are regulated by covalent modifications by fatty acids myristate and palmate. These lipid modifications serve to anchor the subunits to the membrane and increase the interaction with other protein and also increase the affinity of the a-subunit for 3y. In this regard, the myristoylation of Gia is required for adenylyl cyclase inhibition in cell-free assay (Taussig et al. 1993). 

Aroylindoles bind to the colchicine site of p-tubulin, however, and in contrast to colchicine (1), vincristine (2a), nocodazole (13) or taxol (3), they showed no significant influence on the GTPase activity up to 10 pM [48,49]. In addition, angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay was inhibited by D-64131 and analogue. Since 2-aroylindoles are easy to synthesise (Scheme 2), the role of the indole substitution pattern was studied in more detail. 

Randazzo, P. A., Miura, K., and Jackson, T. R. (2001). Assay and purification of phosphoinositide-dependent ADP-ribosylation factor (ARF) GTPase activating proteins. Regulators and effectors of small GTPases. Meth. Enzymol. 329, 343-354. 

Benard, V., and Bokoch, G. M. (2002). Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol. 345, 349-359. 

Fig. 3. In vitro Arf-GTPas assay to measure the GAP activity of ASAPl. (A) The stoichiometry of ASAPl tyrosine phosphorylation can be increased by in vitro kinase reaction performed with purified Flag-ASAP1/Pyk2 complexes. This increase is followed by Western blotting with an anti-phosphotyrosine antibody (pY) and levels of Hag-ASAPl are controlled with an anti-Flag antibody. (B) Arf-GTPase activities of nonphosphorylated (left panel) and phosphorylated ASAPl (right panel) are monitored in a fluorimetric Arfl-GTPase assay as a decrease of the intrinsic Arfl tryptophan fluorescence at 340 nm (in arbitrary units AU) upon excitation at 297.5 nm. Black dots indicate sample prior and grey dots after in vitro kinase reaction.

Robust Colorimetric Assays for Dynamin s Basal and Stimulated GTPase Activities... 

Shpetner and Vallee, 1992 Warnock et al, 1996). We believe that these reported ranges of dynamin s GTPase activity reflect, in part, differences in assay conditions and assembly templates. 

Here we describe a simple, colorimetric assay to measure GTP hydrolysis by dynamin and discuss some of the variables that can affect measurements of dynamin s basal and assembly-stimulated GTPase activity. 

Liposome-stimulated GTPase assays are performed as described for basal except that the final concentration of dynamin used in the assay is lower (typically 0.1-0.5 iiM). Liposomes are added from a 20x stock solution to the dynamin in assay buffer just prior to mixing this 2x dyna-min-liposome stock with an equal volume of the 2x GTP stock in assay buffer to initiate the incubation. The remainder of the assay is as described above except that time points are taken more frequently and typically over a 0-15 min time course. The data in Fig. 2A shows that dynamin s GTPase activity could be stimulated > 100-fold upon assembly onto a liposome template composed of DOPC PI4,5P2 cholesterol (80 15 5 mol%), with maximum stimulation occurring at >150 /iM lipid (Fig. 2A). As previously described (Tuma and Collins, 1994), the concentration dependence for dynamin was sigmoidal, indicating positive cooperativity at low concentrations of dynamin (Fig. 2B). 

The degree of stimulation of dynamin s GTPase activity depends on the composition of the liposomes. The data in Fig. 3A shows that dynamin s GTPase activity is stimulated 100-fold when assayed in the presence of liposomes composed of DOPS, but only 10-fold in the presence of liposomes composed of DOPC PI4,5P2 (90 10 mol%), although dynamin was able to tubulate hposomes of both compositions (Fig. 3B). While we have... 

Fig. 2. Dynamin s GTPase activity is stimulated by assembly onto PI4,5P2-containing liposomes. (A) Dependence of the rate of GTP hydrolysis by dynamin on the concentration of liposomes, assayed at fixed concentrations of dynamin (0.5 jxM) and GTP (1 mM). (B) Dependence of the rate of GTP hydrolysis by dynamin on the concentration of dynamin in the assayed at fixed concentrations of liposomes (25 M) and GTP (1 mM). Note the sigmoidal shape of the curve indicating cooperativity.

Fig. 3. Dynamin s liposome-stimulated GTPase activity varies with liposome composition. (A) Dynamin s GTPase activity is greater when assayed in the presence of liposomes composed exclusively of DOPS, compared to liposomes prepared from DOPC PI4,5P2 (90 10 mol%). Liposomes composed of a higher mol% PI4,5P2 (see Fig. 2) are significantly more effective templates. (B) Negative-stain electron micrographs of dynamin-generated lipid tubules formed on DOPS and DOPC PI4,5P2 liposomes are indistinguishable, despite the observed differences in rates of GTP hydrolysis. Scale bar = 50 nm.

Fig. 1. Dynamin GTPase assay was performed in the presence of truncated amphiphysin. The truncated amphiphysins were designed as shown in (A). Effect of these amphiphysins on dynamin GTPase activity was assayed (B). The data was normalized by the GTPase activity of dynamin. Deletion of the middle domain of amphiphysin (Amph A248-315 and Amph A248-601) resulted in strong stimulation of the dynamin GTPase activity. Binding sites for AP-2 and clathrin are depicted in (A). (Reproduced with permission from Y. Yoshida et al. [2004]. EMBO J. 23, 3483-3491.)...

Dynamin GTPase activity was measured essentially by the method of Barylko (2001). GTPase assay was performed in cytosolic buffer (25 mM Hepes-KOH, pH 7.2, 25 mM KCl, 2.5 mM magnesium acetate, 100 mM potassium glutamate) in 2.0 ml microcentrifuge tubes. One hundred /ul of reaction mixture contained protein and lipid at the following concentrations. 

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