Figure 2.

Effects of NEM on kinesin release from vesicles during homogenization. (a) Microsomal vesicles were purified by homogenizing fresh bovine brains either with or without 2 mM NEM. Three fractions were defined: the 39,800 × g pellet (V1), the 260,000 × g pellet (V2), and the 260,000 × g supernatant (S). The supernatant and vesicle fractions were probed for the presence of kinesin using the H2 antibody on quantitative immunoblots as described previously (Pfister et al., 1989b). The presence of 2 mM NEM during homogenization minimized kinesin release from vesicles during homogenization. Error bars represent ±SEM; n = 3. (b) The concentration dependence of NEM effects on the amount of kinesin in V2 was assayed by varying the concentrations of NEM added to homogenization buffer from 0.1 to 5.0 mM before the homogenization. Kinesin was quantitated as described above. NEM at concentrations of 1–2 mM maximally inhibited kinesin release from vesicles.