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. Author manuscript; available in PMC: 2009 Jan 18.
Published in final edited form as: ACS Chem Biol. 2008 Jul 18;3(7):419–428. doi: 10.1021/cb800083a

Figure 1.

Figure 1

Hydrolyzed DIDS mixture inhibits ClC-ec1 better than freshly prepared DIDS. a) Reversed-phase HPLC chromatogram of a freshly prepared solution of DIDS (top) or hydrolyzed DIDS (bottom). The numbers above each peak represent the fraction numbers referred to throughout the paper. b) Chloride flux assays, measuring the change in extravesicular chloride concentration as a function of time. Triton X-100 was added to determine the total intravesicular chloride concentration. c) Chloride flux through ClC-ec1 was measured at several concentrations of freshly prepared DIDS (closed circles) or hydrolyzed DIDS mix (open squares). The activity (A, defined as the initial rate of chloride efflux through ClC-ec1-containing vesicles minus the initial rate of chloride efflux through control vesicles) is normalized to the activity observed in the absence of inhibitor (Ao). In the right panel, the effects of the hydrolyzed DIDS mix are shown on an expanded scale. Symbols represent the mean activity from four to five assays, and error bars represent the standard error of the mean. Solid lines are fits to the Hill equation: A/Ao = (A/Ao)max/[1 + (K1/2/[Inhibitor])-n] where K1/2 is the apparent affinity and n is the Hill coefficient. For the hydrolyzed DIDS mixture, (A/Ao)max = 1.06, K1/2 = 4.8 μM, and n = 1. For freshly prepared DIDS, (A/Ao)max = 1.08, K1/2 = 300 μM, and n = 2.