| Assay Method Information | |
| | Stopped-flow Assay |
| Description: | Osmotic water permeability was measured at 22-24 °C bymonitoring 90° scattered light intensity at 520 nm wavelength. Measurements were made using a PiStar 180 (Applied Photophysics, UK), with a dead time of 1-2 milliseconds. Thirty minutes prior to the assay, stripped red blood cell ghosts or proteoliposomes containing purified AQP1 and stored in ice were treated with mercuric chloride (50-100 µM; positive control for inhibition) or with the various compounds usually at a concentration of 150 µM. Stock solutions of compounds were in 100% ethanol (for stripped erythrocyte membrane experiments) and in 100% DMSO (for proteoliposome experiments). An inwardly directed osmotic gradient (hyperosmotic shock) was created by mixing the vesicles maintained in Buffer A (50 mM sodium phosphate buffer pH, 100 mM NaCl, 1 mM EDTA, 0.025% sodium azide) with an equal volume of 200 mM mannitol. On the other hand, vesicles preincubated with 200 mM mannitol were mixed with Buffer A to create an outwardly |
| Affinity data for this assay | |
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