After three freeze/thaw cycles, the multilamellar vesicles were extruded 21 times through a 400 nm membrane filter to form proteoliposomes. Liposomes were passed through a 1.5 mL Sephadex G20 resin column equilibrated with 0.3 M potassium isethionate, 1 mM KF, 15 mM HEPES pH 7.0 and diluted 20-fold in assay buffer in a stirred chamber. Fluoride was monitored with a fluoride electrode (Cole Palmer, USA) attached to a pH meter and digitized at a sampling frequency of 5 Hz. Transport was initiated by addition of 1 μM of the potassium ionophore valinomycin to relieve the electrical potential and permit F− and K+ to flow down their chemical gradients. At the end of the experiment, 30 mM octyl-β-D-glucopyranoside was added to release remaining encapsulated fluoride.
Protocol detail
Fluoride Transport Kinetics in Proteoliposomes
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Other organizations : University of Michigan–Ann Arbor
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Variable analysis
independent variables
- Number of freeze/thaw cycles (3)
- Number of extrusions through 400 nm membrane filter (21)
- Fluoride (F-) transport through proteoliposomes
- Remaining encapsulated fluoride after experiment
- Composition of buffer used to equilibrate Sephadex G20 resin column (0.3 M potassium isethionate, 1 mM KF, 15 mM HEPES pH 7.0)
- Dilution factor of liposomes in assay buffer (20-fold)
- Concentration of potassium ionophore valinomycin (1 μM)
- Concentration of octyl-β-D-glucopyranoside used to release remaining encapsulated fluoride (30 mM)
- Positive control: Addition of 1 μM valinomycin to relieve electrical potential and permit F- and K+ flow down their chemical gradients
- Negative control: Not explicitly mentioned
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