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J M Salhany

Publications and source records attributed to J M Salhany.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of immunoglobulin binding to isolated human erythrocyte membranes: evidence for selective, temperature-induced binding of naturally occurring autoantibodies to the cytoskeleton.

Human plasma contains naturally occurring autoantibodies to the predominant components of the erythrocyte membrane: band 3 and spectrin bands 1 and 2 of the cytoskeleton. The titer of cytoskeletal plasma autoantibodies increases in various hemolytic conditions, suggesting that opsonization of the cytoskeleton may play an important role in the clearance of hemolyzed (not senescent) erythrocytes from the circulation. In this study, we use Alexa Fluor 488 goat anti-human IgG conjugate (Molecular Probes, Eugene, OR, USA), to characterize plasma immunoglobulin binding to erythrocyte membranes from osmotically hemolyzed cells ('ghosts'). The results show that exposure of ghosts to plasma results in 4-fold more immunoglobulin binding to the cytoskeleton than is bound to the proteins contained within the lipid bilayer. Preincubation of the ghosts at 37 degrees C causes 8-fold more immunoglobulin binding to the cytoskeleton compared to bilayer proteins. This temperature-induced change resulted from selective immunoglobulin binding to the cytoskeleton, with no change in immunoglobulin binding to bilayer proteins. However, the rate of increase in cytoskeletal antigenicity at 37 degrees C did correlate with the rate of a conformational change in band 3, a transmembrane protein which serves as a major membrane attachment site for the cytoskeleton. The results of this study suggest that the cytoskeleton is the primary target in the opsonization of hemolyzed erythrocyte membranes by naturally occurring plasma autoantibodies. The conformational changes which occur in ghosts at 37 degrees C are associated with selective exposure of new immunoglobulin binding sites on the cytoskeleton, and with a change in the structure of band 3. We propose a model suggesting that opsonization of the cytoskeleton occurs prior to the decomposition of hemolyzed erythrocytes at 37 degrees C.

Autoantibodies↗
PubMed2000

Mechanism of band 3 dimer dissociation during incubation of erythrocyte membranes at 37 degrees C.

The mechanism of dissociation of the stable dimer of band 3 was investigated during the incubation of isolated erythrocyte membranes or resealed ghosts at 37 degrees C. The kinetics of changes in the structural and functional integrity of the membrane domain of band 3 (MDB3) were measured and correlated with the change in the Stokes radius of band 3. MDB3 integrity was determined as follows: (1) by measuring the fluorescence emission spectrum of 4, 4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS) bound covalently to MDB3; (2) by measuring the number of DIDS covalent binding sites present after incubation of unlabelled resealed ghosts; and (3) by measuring the anion transport V(max) by using the same resealed ghosts. Incubation of membranes at 37 degrees C caused the dissociation of band 3 dimers to monomers but only after a lag period lasting approx. 50 h. The observation of such a lag implies that dissociation involves a sequence of molecular events beginning with some type of initial process. We have discovered that this initial process involves a conformation change in MDB3. There was a shift in the fluorescence spectrum for DIDS-labelled band 3 and a decrease in the DIDS binding capacity and transport activity of the unlabelled protein. Incubation of membranes at 4 degrees C inhibited the conformational change in MDB3 and the dissociation of dimers. Furthermore, no conformational change in MDB3 was observed when erythrocytes were incubated at 37 degrees C. We suggest that MDB3 unfolding is the molecular event responsible for the subsequent dissociation of stable dimers of band 3 to monomers during the incubation of erythrocyte membranes at 37 degrees C. The monomers so generated are either not functional in anion exchange or they have an attenuated functionality. The absence of a conformational change for band 3 in erythrocytes might imply that haemolysis perturbs the membrane structure and somehow predisposes band 3 to the conformational change that occurs during incubation at 37 degrees C.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Anion binding characteristics of the band 3 / 4,4-dibenzamidostilbene-2,2-disulfonate binary complex: evidence for both steric and allosteric interactions.

A novel kinetic approach was used to measure monovalent anion binding to better define the mechanistic basis for competition between stilbenedisulfonates and transportable anions on band 3. An anion-induced acceleration in the release of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) from its complex with band 3 was measured using monovalent anions of various size and relative affinity for the transport site. The K1/2 values for anion binding were determined and correlated with transport site affinity constants obtained from the literature and the dehydrated radius of each anion. The results show that anions with ionic radii of 120-200 pm fall on a well-defined correlation line where the ranking of the K1/2 values matched the ranking of the transport site affinity constants (thiocyanate < nitrate approximately bromide < chloride < fluoride). The K1/2 values for the anions on this line were about 4-fold larger than expected for anion binding to inhibitor-free band 3. Such a lowered affinity can be explained in terms of allosteric site-site interactions, since the K1/2 values decreased with increasing anionic size. In contrast, iodide, with an ionic radius of about 212 pm, had a 10-fold lower affinity than predicted by the correlation line established by the smaller monovalent anions. These results indicate that smaller monovalent anions have unobstructed access to the transport site within the band 3 / DBDS binary complex, while iodide experiences significant steric hindrance when binding. The observation of steric hindrance in iodide binding to the band 3 / DBDS binary complex, but not in the binding of smaller monovalent anions, suggests that the stilbenedisulfonate binding site is located at the outer surface of an access channel leading to the transport site.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1998

Characterization of the pH dependence of hemoglobin binding to band 3. Evidence for a pH-dependent conformational change within the hemoglobin-band 3 complex.

The pH dependence of hemoglobin binding to inside-out red cell membrane vesicles was studied using 90 degrees light scattering (Salhany, J.M. et al., Biochemistry 19 (1980) 1447-1454). Hyperbolic binding curves were observed for high-affinity hemoglobin binding to the cytoplasmic domain of band 3 (CDB3) within the intact transporter. Analysis of these saturation curves yielded the apparent Kd and the maximum light scattering signal change (DeltaLSmax ). The apparent Kd for hemoglobin binding did not change substantially between pH 5.5 and 7.0, while at pH 8, there is no binding. In contrast, DeltaLSmax decreased by about 20-fold between pH 5.5 and 7.0, with an apparent pK of 6.5. These results suggest that hemoglobin binds to CDB3 with high affinity at both neutral and acid pH, a suggestion that was confirmed using a centrifugation method. Thus, the pK for the light scattering signal change is significantly lower than the pK for the actual binding process. We show that the change in DeltaLSmax is not related to a change in band 3 binding capacity, which remains constant as a function of pH. We also show that hemoglobin binding to non-band 3 sites contributes less than 10% to DeltaLSmax under our specific experimental conditions. On the basis of these and previous fluorescence quenching results in the literature, we propose a new model for hemoglobin binding to band 3, where raising the pH from 6 and 7 causes the CDB3-hemoglobin complex to undergo a conformational change leading to the movement of the bound hemoglobin away from the surface of the bilayer. The possible implication of this new mechanistic interpretation is discussed briefly.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1998

Spectroscopic and kinetic characterization of eosin-5-maleimide.

Eosin-5-maleimide (EM) is an increasingly important and widely used probe in the study of membrane protein structure and function. Yet little is known about its spectral properties in hydrophobic and hydrophilic environments. Furthermore, EM is hydrolyzed faster than the traditional N-ethylmaleimide. To offer a more solid foundation for the use of EM in studies of membrane protein structure and function, we have undertaken a detailed study of the absorbance and fluorescence spectra of EM, eosin-5-maleic acid, and the L-cysteine and beta-mercaptoethanol adducts of EM in aqueous and hydrophobic environments; we have studied the kinetics of hydrolysis of EM in various environments, and we have investigated the reaction kinetics of EM with L-cysteine.

Cysteine↗
PubMed1998

A novel rapid-reaction spectrophotometric method for monitoring monovalent anion exchange by human erythrocyte band 3.

Thiocyanate (SCN-) uptake into human erythrocytes and resealed ghosts was measured by monitoring the intracellular reaction of SCN- with methemoglobin using a dual wavelength stopped-flow apparatus. The cellular reaction was considerably slower than the reaction of SCN- with methemoglobin in solution, indicating that SCN- diffusion and not chemical reaction was rate limiting. This view was confirmed by showing that the uptake rate followed saturation kinetics (K(m) approximately 9 mM), thus indicating that SCN- transport involves a facilitated diffusion process. Addition of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) totally inhibited SCN- uptake, thus identifying band 3 as the sole facilitator. Substitution of iodide or sulfate for trans chloride, slowed SCN- uptake by 4-fold and 35-fold respectively. Reducing the trans chloride concentration from 150 to 2 mM decreased the extent of the reaction, and slowed the observed rate by about 2-fold. These results define a new approach for the continuous monitoring of monovalent anion exchange by human erythrocyte band 3.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1998

Mechanism of competition between chloride and stilbenedisulfonates for binding to human erythrocyte band 3 (AE1).

Stilbenedisulfonates (S) constitute an important class of competitive inhibitors of the anion exchange (AE) function found in plasma membranes of various cell types. I present a brief summary of recent kinetic studies that provide insight into the mechanism of stilbenedisulfonate-chloride competition in binding to human erythrocyte band 3 (AE1) (B), the chloride-bicarbonate exchanger. Reversible stilbenedisulfonate binding follows a two-step mechanism (S + B <--> SB <--> SB*). Several lines of evidence are summarized that show that chloride, stilbenedisulfonates, and band 3 form a ternary complex, with chloride lowering stilbenedisulfonate affinity allosterically, by accelerating the rate of stilbenedisulfonate release. Of particular significance was our evidence demonstrating that extracellular chloride could accelerate stilbenedisulfonate release from its binding site on the outer surface of band 3 in resealed ghosts (i.e., acceleration in the release of a bound competitive inhibitor by a cis substrate). I suggest that the latter result may be consistent with our earlier proposal that band 3 follows a two-site ordered sequential mechanism, where two allosterically linked chloride binding transport sites move back and forth across the membrane together.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1996

Characterization of the stilbenedisulphonate binding site on band 3 Memphis variant II (Pro-854-->Leu).

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56-->Glu within the cytoplasmic domain, and Pro-854-->Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4'-di-isothiocyanatodihydro-2, 2'-stilbenedisulphonate) to 'lysine A' (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS 'off' rate constant, but no difference was found between mutant and control when DBDS (4,4'-dibenzamido-2,2'-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) 'off' rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4'-di-isothiocyanato-2,2'-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854-->Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

Interactions between mutant and wild-type band 3 subunits in hereditary Southeast Asian ovalocytic red blood cell membranes.

Red cell membranes from individuals with Southeast Asian ovalocytosis (SAO) contain approximately equal proportions of wild-type band 3 and a mutant SAO band 3 which lacks residues 400-408. It is known that the Vmax for anion exchange in SAO cells is reduced by about 50%, that SAO band 3 does not transport anions when expressed alone in a cellular expression system, that SAO band 3 does not bind stilbenedisulfonates, and that about 50% of the band 3 exists as wild-type/SAO heterodimers. In this report, we show that the kinetics of H2DIDS (4,4'-diisothiocyanatodihydro-2,2'-stilbenedisulfonate) release from the wild-type band 3 in SAO membranes is biphasic. The two phases were present in about equal proportions, with rate constants differing by about 5-fold. In contrast; control cells showed monophasic, exponential kinetics with a rate constant comparable to that of the fast phase of SAO membranes. We assign the fast phase in SAO membranes to H2DIDS release from wild-type subunits within homodimers and the slow phase to H2DIDS release from the wild-type subunit within the heterodimer. No differences were observed in kinetic studies of H2DIDS binding. These results suggest that the mutant band 3 subunit alters the conformation of its neighboring wild-type subunit within the heterodimer, resulting in about a 4-fold higher H2DIDS affinity. Additional evidence suggesting that the interactions in the heterodimer may be confined to a region of the wild-type subunit containing the C-terminal subdomain is presented. The relationship of these subunit interactions to the observation of a reduced cellular anion transport function is discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

Allosteric effects in stilbenedisulfonate binding to band 3 protein (AE1).

Stilbenedisulfonates are potent competitive inhibitors of the anion exchange (AE) class of transporters. Although these molecules have been extensively used in studies of the anion exchange function, the actual mechanism by which stilbenedisulfonates compete with transported anions has been uncertain. Over the last several years, work in my laboratory has focused on understanding the mechanism of stilbenedisulfonate binding to human erythrocyte band 3 (AE1), with particular emphasis placed on deciding whether stilbenedisulfonates are pure competitive inhibitors, or whether they inhibit transport allosterically. I summarize our results suggesting that stilbenedisulfonates are allosteric inhibitors of band 3 anion exchange. I also summarize results which show that covalent binding of stilbenedisulfonates to one subunit produces allosteric effects which extend to the neighboring subunit in a band 3 dimer. Such allosteric subunit interactions have been observed: a) in divalent anion influx exchange experiments; b) in reversible stilbenedisulfonate binding studies, and c) in thermal unfolding studies of the membrane domain of band 3. In addition, two quaternary conformational states of the band 3 dimer, modulated by ligands of the stilbenedisulfonate site, have been identified in protein crosslinking studies. Finally, new evidence is discussed showing that Southeast Asian ovalocytic band 3 in a heterodimer composed of mutant and wild-type subunits, increases the 4,4'-diisothiocyanodihydro-2,2'-stilbenedisulfonate (H2DIDS) affinity of the wild-type subunit. Taken together, these results challenge the view that band 3 exists as structurally independent monomers. In addition, they suggest that subunit interactions may play a significant role in the transport function.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868-->Leu).

Band 3 HT (Pro-868-->Leu) is a mutant anion exchange protein which has several phenotypic characteristics, including a 2- to 3-fold larger Vmax, and reduced covalent binding of the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS). We have used fluorescence kinetic methods to study inhibitor binding to band 3 to determine if the point mutation in band 3 HT produces localized or wide-spread conformational changes within the membrane-bound domain of this transporter. Our results show that covalent binding of H2DIDS by band 3 HT is slower by a factor of 10 to 20 compared with the wild-type protein. In contrast, no such difference in the kinetics was observed for covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In addition, the kinetics of H2DIDS release from band 3 HT was abnormal, while the kinetics of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) release showed no difference when compared with the wild-type protein. We conclude that substitution of leucine for proline at position 868 does not perturb the structure of "lysine A" in the membrane-bound domain of band 3 but rather produces an apparently localized conformational change in the C-terminal subdomain of the protein which alters H2DIDS affinity. When combined with the observation of an increased Vmax, these results suggest that protein structural changes at position 868 influence a turnover step in the transport cycle.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Characterization of the stilbenedisulfonate binding site on band 3.

Stilbenedisulfonates are potent inhibitors of Band 3 mediated anion exchange. They bind tightly to the protein and form a 1-to-1 reversible complex. Those stilbenedisulfonates which contain isothocyanato groups such as DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) and H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) can also react rapidly with lysine residues within the binding pocket to yield an irreversible covalent adduct. The reactive lysine residue is known as lysine-A, and is thought to have an unusually low pKa. In this report, we characterize the kinetics of DIDS adduct formation with respect to the effect of substrate anions, competitive inhibitory anions, and pH on the rate of covalent adduct formation. We investigate the following: (a) whether stilbenedisulfonates bind to or block access of substrate anions to the transport site; (b) whether the rapidity of the covalent reaction of DIDS at neutral pH is due to a low pKa for lysine-A within the binding pocket; and (c) whether once bound, DIDS and H2DIDS isothiocyanato groups are accessible to reagents. For this latter experiment, we have utilized a newly discovered reaction of the DIDS isothiocyanato groups with azide to test for accessibility. Our results show that substrate anions, DIDS, and Band 3 form a ternary complex. Significantly, the binding of large substrate anions, such as iodide, is not weakened by DIDS to any greater extent than is the binding of smaller substrates such as chloride or fluoride. These results are not consistent with a "partial blockade" hypothesis for the relationship between the stilbenedisulfonate and transport sites. Rather, they support an allosteric site-site interaction hypothesis. Our pH dependence results show that the apparent pKa for the DIDS/lysine-A reaction is greater than 9.26. This is consistent with typical lysine pKa values, and indicates that lysine-A does not have an unusually low pKa. Finally, we show that azide can react with the isothiocyanato groups of DIDS and H2DIDS within their Band 3 complexes, indicating that the stilbenedisulfonate binding site is accessible to solute. These results support a view which suggests that the stilbenedisulfonate site is a superficial inhibitory site on Band 3 which inhibits transport by allosteric interactions within the protein, rather than by either direct or partial blockade of the transport site.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Effect of chloride on the binding kinetics of various stilbenedisulfonates to band 3.

To determine the mechanism of apparent competitive binding of chloride and stilbenedisulfonates ( S ) to band 3 ( B ), we have compared the binding kinetics of three stilbenedisulfonates [ DIDS, 4,4'-diisothiocyanato-2, 2'-stilbenedisulfonate; H2DIDS 4,4'-diisothiocyanodihydro-2, 2'-stilbenedisulfonate and DBDS, 4,4'-dibenzamido-2, 2'-stilbenedisulfonate ] in the absence and presence of 150 mM sodium chloride at constant ionic strength. Biphasic time courses were observed with the fast phase rate constants following second-order kinetics, and the slow phase rate constants following saturation kinetics according to the mechanism: [formula: see text] The results can be understood in terms of the effect of chloride on each of these reaction steps. Chloride increased k1 by about 2-fold, but decreased k-1 8-fold for H2DIDS. Thus, 150 mM chloride increased the initial affinity of H2DIDS by about 19-fold. There was a 3-fold increase in the initial affinity for DIDS, but little or no effect of chloride on the initial affinity of DBDS. There was no effect of chloride on k2, but, previous "off" rate measurements showed that 150 mM chloride increases k-2 about 16-fold for DBDS and about 12-fold for H2DIDS. Taken together, these results indicate that chloride allosterically competes with stilbenedisulfonates for binding to band 3, predominantly by substantially shifting the second isomerization equilibrium to the left.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Kinetic evidence for ternary complex formation and allosteric interactions in chloride and stilbenedisulfonate binding to band 3.

The molecular basis for chloride and stilbenedisulfonate interaction with band 3 was investigated by measuring the kinetics of stilbenedisulfonate release from its complex with the transporter. We found that 150 mM NaCl accelerated the rate of release of DBDS (4,4'-dibenzamidostilbene-2,2'-dibenzamidostilbene-2,2'-disu lfonate) and H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) by more than 10-fold at constant ionic strength. The acceleration effect saturated as a function of chloride concentration. This is an indication of specific binding within a ternary complex involving stilbenedisulfonate, chloride, and band 3. To see if stilbenedisulfonates block an access channel to the transport site, we studied the effect of rapidly mixing DBDS-saturated resealed ghosts with chloride at constant ionic strength and osmotic pressure. Once again, we observe a large, uniform acceleration in the rate of DBDS release. These findings are not consistent with molecular models where stilbenedisulfonates are proposed to block access to a deeper transport site. We suggest that the intramonomeric stilbenedisulfonate site is not located on the chloride transport pathway but rather interacts with the transport site though heterotropic allosteric site-site interactions. On the basis of our kinetic evidence for ternary complex formation and on transport inhibition evidence in the literature showing a linear dependence of KI-app on substrate, we suggest that stilbenedisulfonates are linear mixed-type inhibitors of band 3 anion exchange, not pure competitive inhibitors as has been assumed on the basis of analysis of transport inhibition data alone.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Calorimetric evidence for allosteric subunit interactions associated with inhibitor binding to band 3 transporter.

A calorimetric endotherm occurring at 68 degrees C (the C-transition) has been assigned previously to the integral domain of band 3 and was shown to be shifted to 78 degrees C after covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In this study, we correlate the fractional appearance of the shifted C-transition with the fraction of DIDS bound to the band 3 monomer population. Our results show a distinctly nonlinear correlation plot with the appearance of the shifted C-transition lagging behind DIDS labeling of the band 3 monomer population. The lag suggests that both monomers of a band 3 dimer must be labeled by DIDS in order for the shifted C-transition to appear at 78 degrees C, implying that the thermal unfolding of the integral domain of band 3 is modulated by allosteric interactions between subunits. This is the first in situ structural evidence supporting ligand-mediated subunit interactions within a "carrier"-type transporter protein oligomer.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Inhibition of gp120-CD4 interaction and human immunodeficiency virus type 1 infection in vitro by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate and related compounds were tested for their ability to inhibit gp120-CD4 interaction and human immunodeficiency virus infection in vitro. The results show that pyridoxal 5'-phosphate is a unique CD4 antagonist whose antiviral potency derives from the presence of both lysine-reactive and anionic substituents.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Kinetics of conformational changes associated with inhibitor binding to the purified band 3 transporter. Direct observation of allosteric subunit interactions.

Subunit interaction effects were identified for isolated human erythrocyte band 3, the anion exchanger, by observing both static and stopped-flow kinetic protein fluorescence changes associated with inhibitor binding to the intramonomeric stilbenedisulfonate site. We measured the rate of conformational changes associated with reversible binding of H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate). The rate of H2DIDS release was also measured. As a test for subunit interactions, we studied the effect of partial labeling of the band 3 monomer population with H2DIDS on the equilibrium and kinetics of H2DIDS reversible binding to the remaining monomers. The results showed biphasic kinetics for control band 3, with a pseudo-first-order ligand dependence for the fast phase followed by a slow ligand-independent relaxation. A second-order "on" rate constant for the fast phase was determined to be (1.2 +/- 0.1) x 10(7) M-1 s-1, while the associated "off" rate constant was found to be 1.1 +/- 0.5 s-1. From these kinetic constants, we calculated a Kd value of 95 +/- 50 nM, which is in excellent agreement with the Kd value determined at thermodynamic equilibrium (110 +/- 9 nM). Covalent labeling of 75% of the band 3 monomer population with H2DIDS changed the kinetics of the fast phase, slowing the apparent rate by changing the order of the reaction from pseudo-first-order to zero-order. Partial labeling did not affect the ligand-independent relaxation. Separate measurements of the H2DIDS "off" rate also showed a biphasic time course, with a 20-fold difference in apparent rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗