Figure 3.

ATP- and KCl-Dependent Release of the Translocator from Microtubules

S3 supernatant was incubated with microtubules and AMP-PNP as described in Experimental Procedures. Many contaminating proteins (lane a), but not the translocator, were removed by resuspending the MT pellet in 2 ml of MTG buffer containing 10 mM AMP-PNP, centrifuging the microtubules, and removing the AMP-PNP-containing supernatant. The translocator could then be obtained by washing the pellet with MTG, releasing with 1 ml of MTG plus 10 mM ATP/0.1 M KCl (30 min; 23°C), centrifuging the microtubules, and collecting the supernatant that contains movement-inducing activity (lane b). The final microtubule pellet was then resuspended in 1 ml of MTG (lane c). To define further the conditions that release translocator from microtubules, S3 supernatant (8 ml) was incubated with microtubules and AMP-PNP, and the microtubule pellet was washed as described above. Microtubules were then aliquoted into eight 100 μl samples in MTG containing the indicated amounts of ATP and KCl and incubated for 30 min at 23°C (lanes d–k). The microtubules were pelleted, and 50 μl of each supernatant was run per lane. Lanes a–c are from a different experiment than lanes d–k.