Dynamin preparation

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.

Rat and bovine brain cytosol are prepared as previously described (Miwako et ah, 2003). To prepare a C1/AP2 and dynamin-depleted ammonium sulfate supernatant of bovine brain cytosol, cytosol is brought to 30% (NH2)4S04 by adding saturated (NH2)4S04 while gently stirring at 4°. 

Clathrin and AP2 are purified from microsome extracts as previously described (Manfredi and Bazari, 1987 Smythe et al, 1992). Clathrin is stored in 30 mM Tris, pH 7.0 in aliquots at —80°. AP2 is stored in XTR transport buffer in aliquots at -80°. Recombinant dynamin-1 is prepared as described (Marks et al., 2001). 

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.

Dynamin can be prepared from a variety of different sources, including rat brain, bacteria, or baculovirus-infected insect cells. The purification protocol relies on the affinity of dynamin for the SH3 domain of amphi-physin 2. The protocol yields approximately 0.5 mg of dynamin from each liter of bacterial culture. 

Incubate Amph2 SH3 coated glutathione beads with dynamin containing lysate for 40 min and wash with incubation buffer. Elute dynamin with 1.1 M NaCl, 20 mM PIPES pH6.2,10 mM CaCl2 and dialyze overnight into incubation buffer. Concentrate using a 50 kDa MWCO Vivaspin concentrator. If the preparation is clean, the sample can be concentrated up to and above 0.5 mg/ml without precipitation. 

Lipid Stocks and Buffers. Lipid nanotube formation uses the same equipment as liposome formation. The major difference is the use of NFA-GalCer (Sigma) as a replacement for phosphatidylethanolamine and phosphatidylserine. NFA-GalCer stocks are prepared to 10 mg/ml in chloroform and stored at 80° under argon. The buffer used for the study of dynamin on lipid nanotubles is 135 mM NaCl, 5 mM KCl, 20 mM HEPES pH 7.4,1 mM MgCl2. 

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