Bas ms 2340

To mimic the sample preparation for AFM measurements, 100 μL of proteoliposomes YEG·A was incubated on a freshly cleaved mica surface (V1-grade, Ted Pella Inc.) on ice for 1 h in a water saturated environment. The surface was rinsed 10 times with 50 μL buffer consisting of 10 mM HEPES, 300 mM KAc, 5 mM MgAc₂, pH 7.6. Then, 50 μL of the solution was removed and replaced with solution containing radioactive ATP (3.3 mM [γ−³²P] ATP, 0.908 nCi/μL, PerkinElmer). At predetermined times, a volume of solution above the surface was removed, mixed with 50 mM EDTA to stop the hydrolysis, and spotted on a TLC plate (Millipore Corporation). The plate was developed in 125 mM KH₂PO₄, dried, exposed to a phosphor imaging plate (Fujifilm BAS-MS 2340) in a closed cassette, and then scanned using a phosphor imager (FLA3000). The percent ATP hydrolyzed was estimated from the proportion of total radioactivity that migrated as inorganic phosphate. To evaluate ATPase activity in solution, the aforementioned procedure was carried out in the absence of the mica incubation and rinsing steps. All experiments were replicated at least three times.