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Kinetic Mechanism of DIDS Binding to Band 3 (AE1) in Human Erythrocyte Membranes
Blood Cells, Molecules, and Diseases (2001) 27(5) Sept/Oct: 844 – 849 doi:10.1006/bcmd.2001.0458, available online at http://www.idealibrary.com on
Salhany and Schopfer
Kinetic Mechanism of DIDS Binding to Band 3 (AE1) in Human Erythrocyte Membranes Submitted 07/27/01 (Communicated by J. Hoffman, M.D., 08/28/01)
James M. Salhany1 and Lawrence M. Schopfer1 ABSTRACT: Stilbenedisulfonates (S) are used widely in cell biology as competitive inhibitors of anion exchange, but the mechanism of competition is not resolved. Resolution requires understanding the detailed steps in the reaction of stilbenedisulfonates with various anion-exchange proteins. Studies on the reversible binding of DBDS (4,4⬘-dibenzamido-2,2⬘-stilbenedisulfonate) and H2DIDS (4,4⬘-diisothiocyanatodihydro-2,2⬘-stilbenedisulfonate) to erythrocyte band 3 (B) have shown biphasic kinetic time courses at 25°C. Yet, results for the reversible binding of DIDS (4,4⬘-diisothiocyanato-2,2⬘-stilbenedisulfonate) are controversial. One recent report has shown monophasic kinetics, in experiments performed at 0°C, and at a single, very low concentration of DIDS (0.1 M). Studies are presented which attempt to reconcile these recent findings with the other kinetic data in the literature. We measure the kinetics of DIDS reversible binding to band 3, over a wide DIDS concentration range. In addition, the time course for DIDS binding to band 3 at 0°C is compared with that at 25°C. The results show biphasic binding kinetics at both 0 and 25°C, and they are consistent with expectations for a two-step binding mechanism (S ⫹ B ^ SB ^ SB*). Furthermore, computer-assisted model simulation studies reveal that monophasic DIDS binding kinetics are generated by a two-step mechanism, when calculations are performed at 0.1 M DIDS and 0°C. Under these conditions the initial binding step in the two-step reaction becomes rate limiting. We conclude that the two-step binding mechanism best describes stilbenedisulfonate binding to band 3 and that the observation of monophasic kinetics at low concentrations of DIDS, while valid, is not mechanistically discriminating, since both one-step and two-step mechanisms can yield the same result. © 2001 Academic Press
INTRODUCTION
H2DIDS (4,4⬘-diisothiocyanatodihydro-2,2⬘-stilbenedisulfonate) reversible binding to band 3, follow biphasic kinetic time courses at 25°C (6 –9). The fast phase apparent rate constant follows second-order kinetics, while the slow phase apparent rate constant shows saturation kinetics. Such behavior is consistent with a two-step binding mechanism:
Our current knowledge of the relationship between band 3 structure and function has depended heavily on experimental studies using stilbenedisulfonates (1). These molecules are potent, and apparently competitive inhibitors of anion exchange (2, 3). Yet, kinetic studies show that chloride accelerates the overall rate of DBDS (4,4⬘dibenzamido-2,2⬘-stilbenedisulfonate) binding to band 3 (4 – 6). To understand how substrate anions can accelerate the binding of an apparently competitive inhibitor, it is essential to define the mechanism by which such inhibitors bind to this transporter. Kinetic studies have shown that DBDS and
k2 k1 S ⫹ B ^ (SB) ^ (SB)*, k⫺1 k⫺2
[1]
where, S is stilbenedisulfonate, B is band 3, (SB) is the initial complex, and (SB)* is the transformed complex (4, 5). However, a recent paper by Janas
Correspondence and reprint requests to: James M. Salhany, Department of Internal Medicine, 985290 Nebraska Medical Center, Omaha, NE 68198-5290. Fax: (402) 559-6309. E-mail: [email protected]. 1 Veterans Administration Medical Center and Department of Internal Medicine and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5290. 1079-9796/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved
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Blood Cells, Molecules, and Diseases (2001) 27(5) Sept/Oct: 844 – 849 doi:10.1006/bcmd.2001.0458, available online at http://www.idealibrary.com on
and Janas (10) has shown that the time course for the reversible binding of DIDS (4,4⬘-diisothiocyanato2,2⬘-stilbenedisulfonate) is monophasic, when the reaction with band 3 is measured at one, very low concentration of DIDS (0.1 M), and at 0°C. The results of Janas and Janas (10) raise a major concern for those who wish to interpret anion exchange inhibition findings using various stilbenedisulfonates. They suggest that the fundamental mechanism of stilbenedisulfonate binding to band 3, may not be as general as originally believed (9, 11). Prior to the report by Janas and Janas (10), reversible binding for all stilbenedisulfonates was found to be biphasic. The results with DIDS at 0°C (10), suggest that either DIDS has a fundamentally different binding mechanism, or some type of drastic change in mechanism occurs at 0°C, to yield monophasic kinetics. Yet, this latter possibility seems unlikely, in light of kinetic studies with DBDS at 1°C, where biphasic kinetics pertain at this low temperature (9). In an attempt to reconcile experimental findings for DIDS, with the findings for the other stilbenedisulfonates, we have studied the reversible binding of DIDS over a wide concentration range, at 25°C, in order to establish the mechanism. We then compare the kinetic time course for DIDS binding to band 3 at 0°C, with that at 25°C under otherwise identical conditions. Finally, we perform a computer-assisted simulation study to show what type of time course is generated for a two-step binding mechanism (Eq. [1]) at 0.1 M DIDS, and at 0°C (10).
measured using the stopped-flow settings just described (11). Furthermore, monophasic kinetic plots are observed when the unimolecular dissociation of stilbenedisulfonates from band 3, is measured using the stilbenedisulfonate replacement reaction (11) under otherwise identical conditions to those used in the binding studies. There was no evidence for sample heterogeneity when membranes containing wild-type band 3 were used (11). Thus, our observation of biphasic kinetics is not a stopped-flow artifact. Determination of the kinetics of binding at each DIDS concentration, involved sampling 1000 data points per reaction trace, and signal averaging between 10 and 30 such traces. Visual inspection of the reaction traces showed that they were biphasic. Data fitting to a four-parameter equation, representing the sum of two exponentials (Fig. 1), gave the best fit to the data. Fitting was accomplished using Sigma Plot (Jandel Scientific, San Rafael, CA).
MATERIALS AND METHODS
RESULTS
Stopped-flow fluorescence of DIDS binding to band 3 in unsealed ghosts. Unsealed ghosts were prepared as described previously (7). We followed the quenching of band 3 protein fluorescence which occurs upon complexation with DIDS, by setting the excitation wavelength of the Gibson–Durrum stopped-flow apparatus at 280 nm, and observing the emission through a 315-nm cutoff filter (8). We have shown that the change in fluorescence signal due to reversible binding of DIDS, is linearly related to binding under stoichiometric conditions ([band 3] ⬎ [DIDS]), when
Figure 1A shows a typical plot of time course data for the kinetics of quenching of band 3 protein fluorescence (8) by reversible binding of 2.5 M DIDS, at 25°C. The time course is biphasic, with apparent rate constants and amplitudes given in the legend to Fig. 1. It should be noted that the biphasic character and the value of the rate constants were found not to vary with pH for the forward-flow kinetics of DIDS binding to band 3, between pH 6 and 8 (data not shown). Similar biphasic time courses were obtained over the DIDS concentration range studied in Fig. 1 (2 to
Theoretical calculations. We used the computer simulation program (Chemical Kinetics Simulation, version 1.01) provided by the IBM Almaden Research Center (San Jose, CA) and designed and created by William Hinsberg and Frances Houle. This program can be downloaded from the IBM Almaden Research Center Web site (www.almaden.ibm.com). Equation [1] above was simulated under conditions equivalent to those published by Janas and Janas (10) (for details, see the legend to Fig. 3).
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FIG. 1. Kinetics of DIDS reversible binding to band 3 at 25°C, as determined using stopped-flow fluorescence. (A) Time course for the change in fluorescence when ghosts ([band 3] ⫽ 0.9 M before the mix) were mixed with an equal volume of 5 M DIDS (i.e., 2.5 M after the mix). The experiments were performed in 150 mM NaCl, plus 5 mM sodium phosphate, pH 8, at 25°C. An equation for the sum of two exponentials was fitted to the data in order to determine the apparent rate constants for the two phases: {(⌬F/⌬F total) ⫽ A fexp( ⫺ k f ⴱ t) ⫹ A sexp( ⫺ k s ⴱ t)}, where the subscripts f and s indicate fast and slow, respectively. The values for the amplitudes ( A f and A s) and rate constants (k f and k s) for the fast and slow phases are: A f ⫽ 0.79 ⫾ 0.02, k f ⫽ 6.2 ⫾ 0.4 s⫺1, A s ⫽ 0.18 ⫾ 0.03, k s ⫽ 0.88 ⫾ 0.1 s⫺1. (B) Plot of the apparent fast phase rate constant versus the concentration of DIDS. The slope of this line is k 1 ⫽ 2.3 ⫾ 0.9 ⫻ 10 6 M⫺1 s⫺1. The intercept value is k ⫺ 1 ⫽ 1.4 ⫾ 0.1 s⫺1. (C) Plot of the apparent slow phase rate constant versus the concentration of DIDS. An hyperbolic equation of the form y ⫽ {(k 2 ⫻ [DIDS])/(K 1 ⫹ [DIDS])} ⫹ k ⫺ 2 was fitted to this data. The values of the constants which gave the best fit are K 1 ⫽ 0.6 ⫾ 0.2 M, k 2 ⫽ 0.7 ⫾ 0.3 s⫺1, and k ⫺ 2 ⫽ 0.2 ⫾ 0.2 s⫺1. The value for the overall binding constant K d [ ⫽ K 1 ⴱ K 2 /(1 ⫹ K 2 )] was calculated to be 133 nM, with K 1 and K 2 defined in the text.
0.7 ⫾ 0.3 s⫺1 and k ⫺ 2 of 0.2 ⫾ 0.2 s⫺1 (note that the apparent rate at saturation is equal to k 2 ⫹ k ⫺ 2 ). Because of the low value of K 1 , it was technically impossible to obtain data at sufficiently low concentrations of DIDS to firmly establish the value of k ⫺ 2 . The overall K d can be calculated using these kinetic values for the reversible binding of DIDS to band 3 {K d ⫽ K 1 ⴱ K 2 /(1 ⫹ K 2 ), where K 1 is defined as above, and with K 2 ⫽ k ⫺ 2 /k 2 }. We obtained a K d value of 133 nanomolar at 25°C, which should be considered an approximate value considering the experimental error associated with determining the value of k ⫺ 2 for DIDS. The data in Fig. 1 were taken at 25°C. Janas and Janas (10) took their data at 0°C. We were concerned that lowering the temperature might have affected the fluidity of the red cell membrane, and caused the second phase to disappear. To test for this possibility, we measured the binding of DIDS to band 3 at 0°C, by reacting isolated erythrocyte membranes with 15 M DIDS (Fig. 2). For comparison, we also show this reaction at 25°C, using the same reagents (Fig. 2). The time
25 M). Figure 1B shows a secondary plot of the observed fast phase rate constant as a function of the concentration of DIDS. It is apparent that the fast phase rate is linearly related to DIDS concentration over the whole concentration range. This behavior implies that the fast phase represents the second-order collision of DIDS with a binding site on band 3. Figure 1C shows that the slow phase rate constant has a saturating dependence on DIDS concentration. This kinetic pattern is consistent with a two-step binding mechanism (Eq. [1]) observed for the other stilbenedisulfonate molecules which have been studied (4 –9). We have treated the two reaction phases as kinetically uncoupled reactions (6, 12), since the apparent rates of the two phases differ by ⬎7-fold at any given concentration of DIDS. The second order binding constant (k 1 in Eq. [1]) determined from the slope of the line in Fig. 1B, is 2.3 ⫾ 0.9 ⫻ 106 M⫺1 s⫺1. The intercept of that line with the y-axis gives a value for k ⫺ 1 of 1.4 ⫾ 0.1 s⫺1. Thus, the equilibrium binding constant for the first step, K 1 , is 0.6 ⫾ 0.2 M, where K 1 ⫽ k ⫺ 1 /k 1 (6). The kinetic constants for the slow phase are k 2 ⫽ 846
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Blood Cells, Molecules, and Diseases (2001) 27(5) Sept/Oct: 844 – 849 doi:10.1006/bcmd.2001.0458, available online at http://www.idealibrary.com on
FIG. 2. Plot of the time courses for the reaction of DIDS with band 3 at 25 and 0°C. The reactions were performed under the same solution conditions as those described in Fig. 1, except that the concentration of DIDS was 15 M after mixing. The fitted values for the fast (f) and slow (s) phase amplitudes ( A f and A s), and rate constants (k f and k s) (see equation in legend to Fig. 1) are (25°C) A f ⫽ 0.7 ⫾ 0.02, k f ⫽ 29.6 ⫾ 1.6 s⫺1; A s ⫽ 0.25 ⫾ 0.01; k s ⫽ 1.04 ⫾ 0.07 s⫺1; (0°C) A f ⫽ 0.73 ⫾ 0.005, k f ⫽ 6.58 ⫾ 0.008 s⫺1, A s ⫽ 0.27 ⫾ 0.002; k s ⫽ 0.42 ⫾ 0.005 s⫺1.
course at 0°C, maintained its biphasic character. The values for the apparent rate constants for each phase were reduced at 0°C, as expected. These temperature dependent results are comparable to the findings of Posner and Dix (9), who showed that DBDS binding was biphasic at both 23.5 and 1°C.
version 1.01, from the IBM Almaden Research Center (see Materials and Methods). For rate constants, we chose values from the DIDS binding data measured at 25°C (11), and reduced those values by 4-fold to correspond to a change in temperature from 25°C to 0°C (k 1 ⫽ 3 ⫻ 10 5 M⫺1 s⫺1, k ⫺ 1 ⫽ 0.55 s⫺1, k 2 ⫽ 0.28 s⫺1, and k ⫺ 2 ⫽ 0.028 s⫺1). The 4-fold reduction was based on the generally accepted assumption that reaction rates are reduced 2-fold for every 10°C reduction in temperature. The resulting value of k 1 (i.e., 3 ⫻ 105 M⫺1 s⫺1, Fig. 3) was close that measured by Janas and Janas (10) for k 1 at 0°C (3.72 ⫻ 105 M⫺1 s⫺1). We used concentrations of band 3 (Species B ⫽ 3 ⫻ 10⫺7 M) and of DIDS (Species S ⫽ 1 ⫻ 10⫺7 M) equivalent to those used by Janas and Janas (10). Under the conditions just described, we see that Species S decreases, Species SB* increases, while only a small amount of Species SB is formed (Fig. 3A). The disappearance of S [which is what was measured by Janas and Janas (10)] occurs in a first-order, monophasic manner (Fig.
Theoretical Simulation of a Two-Step Binding Mechanism for DIDS Binding to Band 3 Janas and Janas (10) measured the binding of DIDS to band 3, at 0°C, by mixing intact red cells with tritiated DIDS, separating the supernatant from the cells at various times, and measuring the amount of tritiated DIDS remaining free in the supernatant. Simulation of the two-step mechanism in Eq. [1] reveals that if low enough ligand concentrations are used initially, monophasic time courses will be observed for the decrease in free DIDS concentration during the reaction. Simulations of Eq. [1] were made for conditions equivalent to those used by Janas and Janas (10). We used the program Chemical Kinetics Simulator 847
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Salhany and Schopfer
FIG. 3. Computer-assisted simulations. (A) Simulation was made of the reaction in Eq. [1] of the text (S ⫹ B ^ SB ^ SB*), where S is [DIDS]; B is [band 3]; SB is [the DIDS-band 3 binary complex], and SB* is [the transformed DIDS-band 3 binary complex]. The values of initial concentrations of species ([S] ⫽ 1 ⫻ 10⫺7 M, [B] ⫽ 3 ⫻ 10⫺7 M) were taken from Janas and Janas (10). The rate constants used for this simulation were taken from stopped-flow fluorescence measurement of DIDS binding to band 3 (11), adjusted for the difference in temperature (see the text), and are as follows: k1 ⫽ 3 ⫻ 105 M⫺1 s⫺1, k⫺1 ⫽ 0.55 s⫺1, k2 ⫽ 0.28 s⫺1, k⫺2 ⫽ 0.028 s⫺1. (B) Semilog plot of S ⫺ Sfinal versus time.
3B). This behavior occurs despite the fact that we are performing the simulation using a two-step binding reaction.
benedisulfonate molecule with a site on band 3, to form an initial binary complex. The second step reflects some type of slower change within that binary complex. This latter process could involve a conformational change within the binary complex, as suggested in work with isolated band 3 (8, 14). If reversible binding of DIDS to band 3 follows biphasic kinetics and lowering the temperature to 0°C does not change that pattern, why then did Janas and Janas (10) observe monophasic kinetics? We suggest a simple answer to this question, which focuses on the specific experimental conditions used in the binding studies of Janas and Janas (10). To observe two reaction phases in a mechanism like that in Eq. [1], it is necessary to work at ligand concentrations which cause the rate of the first step (second-order binding) to substantially exceed the rate of the second, pseudo-zero-order step. Under the conditions used by Janas and Janas (10), the concentration of DIDS was about 0.1 M. This is fully 20 times lower than the lowest concentration of DIDS we
DISCUSSION The results of this paper demonstrate that the kinetics of reversible binding of DIDS to band 3 are fundamentally the same as the published results for the other members of this class of anion exchange inhibitors (11, 13). All stilbenedisulfonates which have been studied to date, show biphasic kinetic time courses. Lowering the temperature from 25 to 0°C, does not change that characteristic (9) (Fig. 2). In every case studied, DBDS (6, 9), H2DIDS (7, 11), and DIDS (Fig. 1), the initial fast phase of the reaction followed second-order kinetics (i.e., linear dependence of k fast on inhibitor concentration), while the slow phase showed saturation kinetics. This type of behavior is consistent with a two-step binding mechanism (Eq. [1]) (4 – 6). The first step reflects the physical collision of a stil848
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used to collect the data in Fig. 1. We showed, in Fig. 3, that under the conditions used for the simulation, where the value of k 1 at 0°C was essentially the same as that published by Janas and Janas (10), the reaction is monophasic at 0.1 M DIDS. This occurs because the first step in the reaction becomes rate limiting (Fig. 3). Clearly, at such a low concentration of DIDS, it is impossible to distinguish between one-step and two-step reaction mechanisms, since both can yield the same result. When experiments were performed at sufficiently high concentrations of DIDS, biphasic time courses were observed at 0°C (Fig. 2). We conclude that the binding of DIDS to band 3 follows a two-step mechanism.
5.
6.
7.
8.
9.
ACKNOWLEDGMENTS We thank Karen Cordes and Renee Sloan for assistance. This work was supported by the Medical Research Service of the Veterans Administration.
10.
11.
REFERENCES 1.
Cabantchik, Z. I., and Greger, R. (1992) Chemical probes for anion transporters of mammalian cell membranes. Am. J. Physiol. 262, C803–C827. 2. Shami, Y., Rothstein, A., and Knauf, P. A. (1978) Identification of the Cl⫺ transport site of human red blood cells by kinetic analysis of the inhibitory effector chemical probes. Biochim. Biophys. Acta 508, 357–363. 3. Falke, J. J., Pace, R. J., and Chan, S. I. (1984) Chloride binding to the anion transport site of band 3. A chloride-35 NMR study. J. Biol. Chem. 259, 6472– 6480. 4. Verkman, A. S., Dix, J. A., and Solomon, A. K. (1983)
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13.
14.
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Anion transport inhibitor binding to band 3 in red blood cell membranes. J. Gen. Physiol. 81, 421– 449. Dix, J. A., Verkman, A. S., and Solomon, A. K. (1986) Binding of chloride and disulfonic stilbene transport inhibitor to red cell band 3. J. Membr. Biol. 89, 211– 223. Salhany, J. M., Sloan, R. L., Cordes, K. A., and Schopfer, L. M. (1995) Quantitative analysis of the kinetics of stilbenedisulfonate binding to band 3. Int. J. Biochem. Cell Biol. 27, 953–964. Salhany, J. M. (1995) Effect of chloride on the binding kinetics of various stilbenedisulfonates to band 3. Biochem. Mol. Biol. Int. 36, 1067–1077. Salhany, J. M., Cordes, K. A., and Schopfer, L. M. (1993) Kinetics of conformational changes associated with inhibitor binding to purified band 3 transporter. Direct observation of allosteric subunit interactions. Biochemistry 32, 7413–7420. Posner, R. G., and Dix, J. A. (1985) Temperature dependence of anion transport inhibitor binding to human red cell membranes. Biophys. Chem. 23, 139 – 145. Janas, T., and Janas, T. (2000) Reversible DIDS binding to band 3 protein in human erythrocyte membranes. Mol. Membr. Biol. 17, 109 –115. Salhany, J. M. (1996) Allosteric effects in stilbenedisulfonate binding to band 3 protein (AE1). Cell. Mol. Biol. 42, 1065–1096. Halford, S. E. (1972) Escherichia coli alkaline phosphatase. Relaxation spectra of ligand binding. Biochem. J. 126, 727–738. Salhany, J. M. (2001) Stilbenedisulfonate binding kinetics to band 3 (AE1): Relationship between transport and stilbenedisulfonate binding sites, and role of subunit interactions in transport. Blood Cells Mol. Dis. 27, 127–134. Batenjany, M. M., Mizukami, H., and Salhany, J. M. (1993) Near-UV circular dichroism of band 3. Evidence for intradomain conformational changes, and interdomain interactions. Biochemistry 32, 663– 668.
Salhany and Schopfer
Kinetic Mechanism of DIDS Binding to Band 3 (AE1) in Human Erythrocyte Membranes Submitted 07/27/01 (Communicated by J. Hoffman, M.D., 08/28/01)
James M. Salhany1 and Lawrence M. Schopfer1 ABSTRACT: Stilbenedisulfonates (S) are used widely in cell biology as competitive inhibitors of anion exchange, but the mechanism of competition is not resolved. Resolution requires understanding the detailed steps in the reaction of stilbenedisulfonates with various anion-exchange proteins. Studies on the reversible binding of DBDS (4,4⬘-dibenzamido-2,2⬘-stilbenedisulfonate) and H2DIDS (4,4⬘-diisothiocyanatodihydro-2,2⬘-stilbenedisulfonate) to erythrocyte band 3 (B) have shown biphasic kinetic time courses at 25°C. Yet, results for the reversible binding of DIDS (4,4⬘-diisothiocyanato-2,2⬘-stilbenedisulfonate) are controversial. One recent report has shown monophasic kinetics, in experiments performed at 0°C, and at a single, very low concentration of DIDS (0.1 M). Studies are presented which attempt to reconcile these recent findings with the other kinetic data in the literature. We measure the kinetics of DIDS reversible binding to band 3, over a wide DIDS concentration range. In addition, the time course for DIDS binding to band 3 at 0°C is compared with that at 25°C. The results show biphasic binding kinetics at both 0 and 25°C, and they are consistent with expectations for a two-step binding mechanism (S ⫹ B ^ SB ^ SB*). Furthermore, computer-assisted model simulation studies reveal that monophasic DIDS binding kinetics are generated by a two-step mechanism, when calculations are performed at 0.1 M DIDS and 0°C. Under these conditions the initial binding step in the two-step reaction becomes rate limiting. We conclude that the two-step binding mechanism best describes stilbenedisulfonate binding to band 3 and that the observation of monophasic kinetics at low concentrations of DIDS, while valid, is not mechanistically discriminating, since both one-step and two-step mechanisms can yield the same result. © 2001 Academic Press
INTRODUCTION
H2DIDS (4,4⬘-diisothiocyanatodihydro-2,2⬘-stilbenedisulfonate) reversible binding to band 3, follow biphasic kinetic time courses at 25°C (6 –9). The fast phase apparent rate constant follows second-order kinetics, while the slow phase apparent rate constant shows saturation kinetics. Such behavior is consistent with a two-step binding mechanism:
Our current knowledge of the relationship between band 3 structure and function has depended heavily on experimental studies using stilbenedisulfonates (1). These molecules are potent, and apparently competitive inhibitors of anion exchange (2, 3). Yet, kinetic studies show that chloride accelerates the overall rate of DBDS (4,4⬘dibenzamido-2,2⬘-stilbenedisulfonate) binding to band 3 (4 – 6). To understand how substrate anions can accelerate the binding of an apparently competitive inhibitor, it is essential to define the mechanism by which such inhibitors bind to this transporter. Kinetic studies have shown that DBDS and
k2 k1 S ⫹ B ^ (SB) ^ (SB)*, k⫺1 k⫺2
[1]
where, S is stilbenedisulfonate, B is band 3, (SB) is the initial complex, and (SB)* is the transformed complex (4, 5). However, a recent paper by Janas
Correspondence and reprint requests to: James M. Salhany, Department of Internal Medicine, 985290 Nebraska Medical Center, Omaha, NE 68198-5290. Fax: (402) 559-6309. E-mail: [email protected]. 1 Veterans Administration Medical Center and Department of Internal Medicine and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5290. 1079-9796/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved
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and Janas (10) has shown that the time course for the reversible binding of DIDS (4,4⬘-diisothiocyanato2,2⬘-stilbenedisulfonate) is monophasic, when the reaction with band 3 is measured at one, very low concentration of DIDS (0.1 M), and at 0°C. The results of Janas and Janas (10) raise a major concern for those who wish to interpret anion exchange inhibition findings using various stilbenedisulfonates. They suggest that the fundamental mechanism of stilbenedisulfonate binding to band 3, may not be as general as originally believed (9, 11). Prior to the report by Janas and Janas (10), reversible binding for all stilbenedisulfonates was found to be biphasic. The results with DIDS at 0°C (10), suggest that either DIDS has a fundamentally different binding mechanism, or some type of drastic change in mechanism occurs at 0°C, to yield monophasic kinetics. Yet, this latter possibility seems unlikely, in light of kinetic studies with DBDS at 1°C, where biphasic kinetics pertain at this low temperature (9). In an attempt to reconcile experimental findings for DIDS, with the findings for the other stilbenedisulfonates, we have studied the reversible binding of DIDS over a wide concentration range, at 25°C, in order to establish the mechanism. We then compare the kinetic time course for DIDS binding to band 3 at 0°C, with that at 25°C under otherwise identical conditions. Finally, we perform a computer-assisted simulation study to show what type of time course is generated for a two-step binding mechanism (Eq. [1]) at 0.1 M DIDS, and at 0°C (10).
measured using the stopped-flow settings just described (11). Furthermore, monophasic kinetic plots are observed when the unimolecular dissociation of stilbenedisulfonates from band 3, is measured using the stilbenedisulfonate replacement reaction (11) under otherwise identical conditions to those used in the binding studies. There was no evidence for sample heterogeneity when membranes containing wild-type band 3 were used (11). Thus, our observation of biphasic kinetics is not a stopped-flow artifact. Determination of the kinetics of binding at each DIDS concentration, involved sampling 1000 data points per reaction trace, and signal averaging between 10 and 30 such traces. Visual inspection of the reaction traces showed that they were biphasic. Data fitting to a four-parameter equation, representing the sum of two exponentials (Fig. 1), gave the best fit to the data. Fitting was accomplished using Sigma Plot (Jandel Scientific, San Rafael, CA).
MATERIALS AND METHODS
RESULTS
Stopped-flow fluorescence of DIDS binding to band 3 in unsealed ghosts. Unsealed ghosts were prepared as described previously (7). We followed the quenching of band 3 protein fluorescence which occurs upon complexation with DIDS, by setting the excitation wavelength of the Gibson–Durrum stopped-flow apparatus at 280 nm, and observing the emission through a 315-nm cutoff filter (8). We have shown that the change in fluorescence signal due to reversible binding of DIDS, is linearly related to binding under stoichiometric conditions ([band 3] ⬎ [DIDS]), when
Figure 1A shows a typical plot of time course data for the kinetics of quenching of band 3 protein fluorescence (8) by reversible binding of 2.5 M DIDS, at 25°C. The time course is biphasic, with apparent rate constants and amplitudes given in the legend to Fig. 1. It should be noted that the biphasic character and the value of the rate constants were found not to vary with pH for the forward-flow kinetics of DIDS binding to band 3, between pH 6 and 8 (data not shown). Similar biphasic time courses were obtained over the DIDS concentration range studied in Fig. 1 (2 to
Theoretical calculations. We used the computer simulation program (Chemical Kinetics Simulation, version 1.01) provided by the IBM Almaden Research Center (San Jose, CA) and designed and created by William Hinsberg and Frances Houle. This program can be downloaded from the IBM Almaden Research Center Web site (www.almaden.ibm.com). Equation [1] above was simulated under conditions equivalent to those published by Janas and Janas (10) (for details, see the legend to Fig. 3).
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FIG. 1. Kinetics of DIDS reversible binding to band 3 at 25°C, as determined using stopped-flow fluorescence. (A) Time course for the change in fluorescence when ghosts ([band 3] ⫽ 0.9 M before the mix) were mixed with an equal volume of 5 M DIDS (i.e., 2.5 M after the mix). The experiments were performed in 150 mM NaCl, plus 5 mM sodium phosphate, pH 8, at 25°C. An equation for the sum of two exponentials was fitted to the data in order to determine the apparent rate constants for the two phases: {(⌬F/⌬F total) ⫽ A fexp( ⫺ k f ⴱ t) ⫹ A sexp( ⫺ k s ⴱ t)}, where the subscripts f and s indicate fast and slow, respectively. The values for the amplitudes ( A f and A s) and rate constants (k f and k s) for the fast and slow phases are: A f ⫽ 0.79 ⫾ 0.02, k f ⫽ 6.2 ⫾ 0.4 s⫺1, A s ⫽ 0.18 ⫾ 0.03, k s ⫽ 0.88 ⫾ 0.1 s⫺1. (B) Plot of the apparent fast phase rate constant versus the concentration of DIDS. The slope of this line is k 1 ⫽ 2.3 ⫾ 0.9 ⫻ 10 6 M⫺1 s⫺1. The intercept value is k ⫺ 1 ⫽ 1.4 ⫾ 0.1 s⫺1. (C) Plot of the apparent slow phase rate constant versus the concentration of DIDS. An hyperbolic equation of the form y ⫽ {(k 2 ⫻ [DIDS])/(K 1 ⫹ [DIDS])} ⫹ k ⫺ 2 was fitted to this data. The values of the constants which gave the best fit are K 1 ⫽ 0.6 ⫾ 0.2 M, k 2 ⫽ 0.7 ⫾ 0.3 s⫺1, and k ⫺ 2 ⫽ 0.2 ⫾ 0.2 s⫺1. The value for the overall binding constant K d [ ⫽ K 1 ⴱ K 2 /(1 ⫹ K 2 )] was calculated to be 133 nM, with K 1 and K 2 defined in the text.
0.7 ⫾ 0.3 s⫺1 and k ⫺ 2 of 0.2 ⫾ 0.2 s⫺1 (note that the apparent rate at saturation is equal to k 2 ⫹ k ⫺ 2 ). Because of the low value of K 1 , it was technically impossible to obtain data at sufficiently low concentrations of DIDS to firmly establish the value of k ⫺ 2 . The overall K d can be calculated using these kinetic values for the reversible binding of DIDS to band 3 {K d ⫽ K 1 ⴱ K 2 /(1 ⫹ K 2 ), where K 1 is defined as above, and with K 2 ⫽ k ⫺ 2 /k 2 }. We obtained a K d value of 133 nanomolar at 25°C, which should be considered an approximate value considering the experimental error associated with determining the value of k ⫺ 2 for DIDS. The data in Fig. 1 were taken at 25°C. Janas and Janas (10) took their data at 0°C. We were concerned that lowering the temperature might have affected the fluidity of the red cell membrane, and caused the second phase to disappear. To test for this possibility, we measured the binding of DIDS to band 3 at 0°C, by reacting isolated erythrocyte membranes with 15 M DIDS (Fig. 2). For comparison, we also show this reaction at 25°C, using the same reagents (Fig. 2). The time
25 M). Figure 1B shows a secondary plot of the observed fast phase rate constant as a function of the concentration of DIDS. It is apparent that the fast phase rate is linearly related to DIDS concentration over the whole concentration range. This behavior implies that the fast phase represents the second-order collision of DIDS with a binding site on band 3. Figure 1C shows that the slow phase rate constant has a saturating dependence on DIDS concentration. This kinetic pattern is consistent with a two-step binding mechanism (Eq. [1]) observed for the other stilbenedisulfonate molecules which have been studied (4 –9). We have treated the two reaction phases as kinetically uncoupled reactions (6, 12), since the apparent rates of the two phases differ by ⬎7-fold at any given concentration of DIDS. The second order binding constant (k 1 in Eq. [1]) determined from the slope of the line in Fig. 1B, is 2.3 ⫾ 0.9 ⫻ 106 M⫺1 s⫺1. The intercept of that line with the y-axis gives a value for k ⫺ 1 of 1.4 ⫾ 0.1 s⫺1. Thus, the equilibrium binding constant for the first step, K 1 , is 0.6 ⫾ 0.2 M, where K 1 ⫽ k ⫺ 1 /k 1 (6). The kinetic constants for the slow phase are k 2 ⫽ 846
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Blood Cells, Molecules, and Diseases (2001) 27(5) Sept/Oct: 844 – 849 doi:10.1006/bcmd.2001.0458, available online at http://www.idealibrary.com on
FIG. 2. Plot of the time courses for the reaction of DIDS with band 3 at 25 and 0°C. The reactions were performed under the same solution conditions as those described in Fig. 1, except that the concentration of DIDS was 15 M after mixing. The fitted values for the fast (f) and slow (s) phase amplitudes ( A f and A s), and rate constants (k f and k s) (see equation in legend to Fig. 1) are (25°C) A f ⫽ 0.7 ⫾ 0.02, k f ⫽ 29.6 ⫾ 1.6 s⫺1; A s ⫽ 0.25 ⫾ 0.01; k s ⫽ 1.04 ⫾ 0.07 s⫺1; (0°C) A f ⫽ 0.73 ⫾ 0.005, k f ⫽ 6.58 ⫾ 0.008 s⫺1, A s ⫽ 0.27 ⫾ 0.002; k s ⫽ 0.42 ⫾ 0.005 s⫺1.
course at 0°C, maintained its biphasic character. The values for the apparent rate constants for each phase were reduced at 0°C, as expected. These temperature dependent results are comparable to the findings of Posner and Dix (9), who showed that DBDS binding was biphasic at both 23.5 and 1°C.
version 1.01, from the IBM Almaden Research Center (see Materials and Methods). For rate constants, we chose values from the DIDS binding data measured at 25°C (11), and reduced those values by 4-fold to correspond to a change in temperature from 25°C to 0°C (k 1 ⫽ 3 ⫻ 10 5 M⫺1 s⫺1, k ⫺ 1 ⫽ 0.55 s⫺1, k 2 ⫽ 0.28 s⫺1, and k ⫺ 2 ⫽ 0.028 s⫺1). The 4-fold reduction was based on the generally accepted assumption that reaction rates are reduced 2-fold for every 10°C reduction in temperature. The resulting value of k 1 (i.e., 3 ⫻ 105 M⫺1 s⫺1, Fig. 3) was close that measured by Janas and Janas (10) for k 1 at 0°C (3.72 ⫻ 105 M⫺1 s⫺1). We used concentrations of band 3 (Species B ⫽ 3 ⫻ 10⫺7 M) and of DIDS (Species S ⫽ 1 ⫻ 10⫺7 M) equivalent to those used by Janas and Janas (10). Under the conditions just described, we see that Species S decreases, Species SB* increases, while only a small amount of Species SB is formed (Fig. 3A). The disappearance of S [which is what was measured by Janas and Janas (10)] occurs in a first-order, monophasic manner (Fig.
Theoretical Simulation of a Two-Step Binding Mechanism for DIDS Binding to Band 3 Janas and Janas (10) measured the binding of DIDS to band 3, at 0°C, by mixing intact red cells with tritiated DIDS, separating the supernatant from the cells at various times, and measuring the amount of tritiated DIDS remaining free in the supernatant. Simulation of the two-step mechanism in Eq. [1] reveals that if low enough ligand concentrations are used initially, monophasic time courses will be observed for the decrease in free DIDS concentration during the reaction. Simulations of Eq. [1] were made for conditions equivalent to those used by Janas and Janas (10). We used the program Chemical Kinetics Simulator 847
Blood Cells, Molecules, and Diseases (2001) 27(5) Sept/Oct: 844 – 849 doi:10.1006/bcmd.2001.0458, available online at http://www.idealibrary.com on
Salhany and Schopfer
FIG. 3. Computer-assisted simulations. (A) Simulation was made of the reaction in Eq. [1] of the text (S ⫹ B ^ SB ^ SB*), where S is [DIDS]; B is [band 3]; SB is [the DIDS-band 3 binary complex], and SB* is [the transformed DIDS-band 3 binary complex]. The values of initial concentrations of species ([S] ⫽ 1 ⫻ 10⫺7 M, [B] ⫽ 3 ⫻ 10⫺7 M) were taken from Janas and Janas (10). The rate constants used for this simulation were taken from stopped-flow fluorescence measurement of DIDS binding to band 3 (11), adjusted for the difference in temperature (see the text), and are as follows: k1 ⫽ 3 ⫻ 105 M⫺1 s⫺1, k⫺1 ⫽ 0.55 s⫺1, k2 ⫽ 0.28 s⫺1, k⫺2 ⫽ 0.028 s⫺1. (B) Semilog plot of S ⫺ Sfinal versus time.
3B). This behavior occurs despite the fact that we are performing the simulation using a two-step binding reaction.
benedisulfonate molecule with a site on band 3, to form an initial binary complex. The second step reflects some type of slower change within that binary complex. This latter process could involve a conformational change within the binary complex, as suggested in work with isolated band 3 (8, 14). If reversible binding of DIDS to band 3 follows biphasic kinetics and lowering the temperature to 0°C does not change that pattern, why then did Janas and Janas (10) observe monophasic kinetics? We suggest a simple answer to this question, which focuses on the specific experimental conditions used in the binding studies of Janas and Janas (10). To observe two reaction phases in a mechanism like that in Eq. [1], it is necessary to work at ligand concentrations which cause the rate of the first step (second-order binding) to substantially exceed the rate of the second, pseudo-zero-order step. Under the conditions used by Janas and Janas (10), the concentration of DIDS was about 0.1 M. This is fully 20 times lower than the lowest concentration of DIDS we
DISCUSSION The results of this paper demonstrate that the kinetics of reversible binding of DIDS to band 3 are fundamentally the same as the published results for the other members of this class of anion exchange inhibitors (11, 13). All stilbenedisulfonates which have been studied to date, show biphasic kinetic time courses. Lowering the temperature from 25 to 0°C, does not change that characteristic (9) (Fig. 2). In every case studied, DBDS (6, 9), H2DIDS (7, 11), and DIDS (Fig. 1), the initial fast phase of the reaction followed second-order kinetics (i.e., linear dependence of k fast on inhibitor concentration), while the slow phase showed saturation kinetics. This type of behavior is consistent with a two-step binding mechanism (Eq. [1]) (4 – 6). The first step reflects the physical collision of a stil848
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used to collect the data in Fig. 1. We showed, in Fig. 3, that under the conditions used for the simulation, where the value of k 1 at 0°C was essentially the same as that published by Janas and Janas (10), the reaction is monophasic at 0.1 M DIDS. This occurs because the first step in the reaction becomes rate limiting (Fig. 3). Clearly, at such a low concentration of DIDS, it is impossible to distinguish between one-step and two-step reaction mechanisms, since both can yield the same result. When experiments were performed at sufficiently high concentrations of DIDS, biphasic time courses were observed at 0°C (Fig. 2). We conclude that the binding of DIDS to band 3 follows a two-step mechanism.
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ACKNOWLEDGMENTS We thank Karen Cordes and Renee Sloan for assistance. This work was supported by the Medical Research Service of the Veterans Administration.
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Cabantchik, Z. I., and Greger, R. (1992) Chemical probes for anion transporters of mammalian cell membranes. Am. J. Physiol. 262, C803–C827. 2. Shami, Y., Rothstein, A., and Knauf, P. A. (1978) Identification of the Cl⫺ transport site of human red blood cells by kinetic analysis of the inhibitory effector chemical probes. Biochim. Biophys. Acta 508, 357–363. 3. Falke, J. J., Pace, R. J., and Chan, S. I. (1984) Chloride binding to the anion transport site of band 3. A chloride-35 NMR study. J. Biol. Chem. 259, 6472– 6480. 4. Verkman, A. S., Dix, J. A., and Solomon, A. K. (1983)
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Anion transport inhibitor binding to band 3 in red blood cell membranes. J. Gen. Physiol. 81, 421– 449. Dix, J. A., Verkman, A. S., and Solomon, A. K. (1986) Binding of chloride and disulfonic stilbene transport inhibitor to red cell band 3. J. Membr. Biol. 89, 211– 223. Salhany, J. M., Sloan, R. L., Cordes, K. A., and Schopfer, L. M. (1995) Quantitative analysis of the kinetics of stilbenedisulfonate binding to band 3. Int. J. Biochem. Cell Biol. 27, 953–964. Salhany, J. M. (1995) Effect of chloride on the binding kinetics of various stilbenedisulfonates to band 3. Biochem. Mol. Biol. Int. 36, 1067–1077. Salhany, J. M., Cordes, K. A., and Schopfer, L. M. (1993) Kinetics of conformational changes associated with inhibitor binding to purified band 3 transporter. Direct observation of allosteric subunit interactions. Biochemistry 32, 7413–7420. Posner, R. G., and Dix, J. A. (1985) Temperature dependence of anion transport inhibitor binding to human red cell membranes. Biophys. Chem. 23, 139 – 145. Janas, T., and Janas, T. (2000) Reversible DIDS binding to band 3 protein in human erythrocyte membranes. Mol. Membr. Biol. 17, 109 –115. Salhany, J. M. (1996) Allosteric effects in stilbenedisulfonate binding to band 3 protein (AE1). Cell. Mol. Biol. 42, 1065–1096. Halford, S. E. (1972) Escherichia coli alkaline phosphatase. Relaxation spectra of ligand binding. Biochem. J. 126, 727–738. Salhany, J. M. (2001) Stilbenedisulfonate binding kinetics to band 3 (AE1): Relationship between transport and stilbenedisulfonate binding sites, and role of subunit interactions in transport. Blood Cells Mol. Dis. 27, 127–134. Batenjany, M. M., Mizukami, H., and Salhany, J. M. (1993) Near-UV circular dichroism of band 3. Evidence for intradomain conformational changes, and interdomain interactions. Biochemistry 32, 663– 668.