GTPase assay

The Sec23 subunit of the Sec23/24 complex is the GAP for the GTPase, Sari (Aridor et al, 2001 Kuge et al, 1994). This property can be used to assess the function of the Sec23/24 complex in an in vitro GTPase assay. 

In Vitro Arf GTPase Assay to Study the Modulation of ASAPl GAP Activity by Tyrosine Phosphorylation... 

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.

We and others have previously described dynamin GTPase assays that... 

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). 

To initiate GTPase assays, 175 /rl of the dynamin/GED mixtures in PCR tubes are transferred to 37° and 25 //I of 4 mM GTP, 5mM MgCl2 in H2O is added and rapidly mixed. A 0 min time point is taken while the mixtures are still on ice by transferring 17.5 //I of dynamin/GED and 2.5 of GTP stock to a microtiter well containing 5 //I EDTA. 20-/ul aliquots are removed for each subsequent time point and the colorimetric determination of Pi released is performed as described above. Additional controls include dynamin alone and GED alone, subjected to the same dialysis (Fig. 5A). 

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. 

Rapid Purification of Native Dynamin I and Colorimetric GTPase Assay... 

SH3 domains. The McMahon lab suggested the combination of the amphi-physin II—SH3 domain and 1.2 M NaCl to elute dynamin (Vallis et al, 1999). We use an extract from 200 g of sheep brain to purify dynamin in 3 days with a recovery of 8-15 mg of protein at high purity (greater than 98%). The purification of 15 mg of dynamin I provides enough sample for about 50,000 GTPase assays (at 0.3 fig dynamin per sample). 

Fig. 2. Examples of the dynamin I colorimetric GTPase assay. (A) L-a-phosphatidyl-L-serine (PS) liposome concentration curve. Stimulation of dynamin I GTPase activity was measured as a function of PS concentration using the colorimetric assay. The curve reaches a plateau at about 30 /xg/ml PS in this experiment, but varies between PS preparations. The curve is representative of the level of optimal dynamin activity in the absorbance range 0.4-0.6 OD units. (B) Concentration-dependent effect of compound A on dynamin I GTPase activity stimulated by 40 /xg/ml PS. The effect of the drug alone at increasing concentrations is shown in the open bars. The stimulation by PS + Compound A is shown in the hatched bars. Compound A reduces PS-stimulated dynamin I GTPase activity (hatched bars), until at high concentrations there appears to be stimulation. The solid bars show a subtraction of the other values, which, after eliminating the effect of background phosphate, reveals the full inhibitory curve of compound A.

Fig. 3. Phosphate standard curve for the malachite green assay. The assay is linear over the range 1-200 iiM phosphate. Maximum sensitivity is limited by the optical density units on the spectrophotometer, which declines above 2.5 units. The inset shows the standard curve for a typical GTPase assay and is linear in the assay sensitivity range. All values are n = 3 and standard deviations are shown, but are normally smaller than the symbols.

Here we describe four assays that monitor auxilin-dynamin interactions. Included are a nucleotide-dependent dynamin affinity column, a coprecipitation assay that measures auxilin-dynamin nucleotide-dependent direct binding, a GTPase assay that examines auxilin s ability to inhibit the stimulated rate of GTP hydrolysis, and FRET with FLIM analysis of dynamin-auxilin interaction in cells. 

A Continuous, Regenerative Coupled GTPase Assay for Dynamln-Related Proteins... 

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