GTPase activity colorimetric assays

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

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. 

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.

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