FIGURE 4.
The NSF hexamer binds 12 ADP molecules at saturation. Purified NSF (20 nm) was incubated at 30 °C with varying concentrations of [α-32P]ATP (0.5–2000 μm) in the presence of Mg2+, and nucleotide·protein complexes were quantified for protein and nucleotide content. NSF binds 11.5 ± 0.8 molecules of [α-32P]ATP per hexamer with a Kd of 20 ± 4.8 μm. With [γ-32P]ATP used in the same binding experiment, no 32P signal is detected (data not shown), indicating that NSF binds 12 ADP molecules at equilibrium.