[References] [Title page]

Figures

figure 1
Figure 1. Dynamic quenching of SPQ by chloride and bicarbonate solutions. The fluorescence of a solution of 500 [micro]M SPQ, 20 mM HEPES, pH 7.4, and varying chloride and bicarbonate concentrations (total anion concentration = 150 mM) was measured at 25 °C. The data represent the average of three experiments. A weighted fit of a linear function to this data gives an apparent quenching constant of 0.064 ± 0.004 mM-1. Four sets of these experiments gave an average quenching constant of 0.071 ± 0.016 mM-1. Inset: Dynamic quenching of SPQ fluorescence by HEPES.
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figure 2
Figure 2. Time course of SPQ fluorescence. SPQ-labeled, resealed ghost membranes (0.2 mg protein/ml) in buffer solution of chloride, bicarbonate and 20 mM HEPES, pH 7.5, were mixed with an equal volume of chloride, bicarbonate and 20 mM HEPES, pH 7.5. The composition of the two solutions was varied to produce varying chloride gradients across the membrane; the sum of chloride and bicarbonate concentrations was 150 mM. From top to bottom, the chloride gradient was -65, -45, -25, +25, +45 mM. (A negative chloride gradient corresponds to an inwardly-directed chloride gradient.) The final equilibrium intracellular and extracellular chloride and bicarbonate concentrations were 75 mM. The data represent the average of six to eight scans. For these and the following figures, SPQ fluorescence is in arbitrary units.
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figure 3a figure 3b
Figure 3. Fit of fluorescence time course. Experiments were performed as in figure 2. (a) Inwardly-directed chloride gradient is -65 mM; equilibrium chloride concentration is 75 mM. A fit of equation 5 to the data (solid line) gives a time constant of 0.74 ± 0.04 s. The graph below the data is the deviation between the experimental data and the fit, expressed as a fraction of the signal amplitude. (b) As in (a), but with outwardly-directed chloride gradient of +65 mM. The fit gives a time constant of 0.69 ± 0.04 s.
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figure 4
Figure 4. Ping-pong mechanism of anion exchange. The translocation steps are slow compared to the anion binding steps. Equilibrium constants are expressed as dissociation constants. The symbol E stands for band 3.
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figure 5a figure 5b
Figure 5. Effect of anion exchange inhibitors on fluorescence time course. (a) Effect of DIDS, an irreversible inhibitor of anion exchange, on chloride influx resulting from a -65 mM chloride gradient. Equilibrium chloride concentration is 75 mM. Red cells were labeled irreversibly with DIDS before hemolysis as described in Materials and Methods. Exchange time constants were 0.73 ± 0.03 s (control) and 62 ±4 s (DIDS). (b) Effect of 4 [micro]M DBDS, a reversible inhibitor of anion exchange, on chloride efflux resulting from a +65 mM chloride gradient; equilibrium chloride concentration is 75 mM. Exchange time constants were 0.65 ± 0.04 s (control) and 6.8 ± 0.6 s (DBDS). DBDS exerts a considerable inner filter effect on SPQ fluorescence, artifactually lowering the equilibrium SPQ fluorescence.
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figure 6
Figure 6. Temperature dependence of anion exchange. The chloride-bicarbonate exchange rate is in s-1. The line is a fit of the Arrhenius equation to the data with activation energy 17 ± 2 kcal/mole. Data were obtained with a chloride gradient of -30 mM.
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figure 7
Figure 7. Concentration dependence of chloride-bicarbonate exchange rate. The exchange rate was determined at each equilibrium chloride concentration with one to three concentration gradients.
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[References] [Title page]