GTPase activity liposome-stimulated assay

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.

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). 

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. 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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