Binding of DTNB to band 3 in the human red cell membrane.

Inhibition of red cell water transport by the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) has been reported by Naccache and Sha'afi ((1974) J. Cell Physiol. 84, 449-456) but other investigators have not been able to confirm this observation. Brown et al. ((1975) Nature 254, 523-525) have shown that, under appropriate conditions, DTNB binds only to band 3 in the red cell membrane. We have made a detailed investigation of DTNB binding to red cell membranes that had been treated with the sulfhydryl reagent N-ethylmaleimide (NEM), and our results confirm the observation of Brown et al. Since this covalent binding site does not react with either N-ethylmaleimide or the sulfhydryl reagent pCMBS (p-chloromercuribenzenesulfonate), its presence has not previously been reported. This covalent site does not inhibit water transport nor does it affect any transport process we have studied. There is an additional low-affinity (non-covalent) DTNB site that Reithmeier ((1983) Biochim. Biophys. Acta 732, 122-125) has shown to inhibit anion transport. In N-ethylmaleimide-treated red cells, we have found that this binding site inhibits water transport and that the inhibition can be partially reversed by the specific stilbene anion exchange transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), thus linking water transport to anion exchange. DTNB binding to this low-affinity site also inhibits ethylene glycol and methyl urea transport with the same KI as that for water inhibition, thus linking these transport systems to that for water and anions. These results support the view that band 3 is a principal constituent of the red cell aqueous channel, through which urea and ethylene glycol also enter the cell.

Interaction of thiourea with band 3 in human red cell membranes.

Although urea transport across the human red cell membrane has been studied extensively, there is disagreement as to whether urea and water permeate the red cell by the same channel. We have suggested that the red cell anion transport protein, band 3, is responsible for both water and urea transport. Thiourea inhibits urea transport and also modulates the normal inhibition of water transport produced by the sulfhydryl reagent, pCMBS. In view of these interactions, we have looked for independent evidence of interaction between thiourea and band 3. Since the fluorescent stilbene anion transport inhibitor, DBDS, increases its fluorescence by two orders of magnitude when bound to band 3 we have used this fluorescence enhancement to study thiourea/band 3 interactions. Our experiments have shown that there is a thiourea binding site on band 3 and we have determined the kinetic and equilibrium constants describing this interaction. Furthermore, pCMBS has been found to modulate the thiourea/band 3 interaction and we have determined the kinetic and equilibrium constants of the interaction in the presence of pCMBS. These experiments indicate that there is an operational complex which transmits conformational signals among the thiourea, pCMBS and DBDS sites. This finding is consistent with the view that a single protein or protein complex is responsible for all the red cell transport functions in which urea is involved.

Kinetics and mechanism of Ca2+ binding to arsenazo III and antipyrylazo III.

Equilibrium and temperature-jump spectrophotometric measurements were carried out on arsenazo III (Ar) and antipyrylazo III (Ap) in order to establish the kinetic reaction schemes for complexing of these dyes with Ca2+. The reaction media contained 30 mM Na2HPO4 as the buffer salt, at pH 7.4. Dependence of the relaxation rate of arsenazo III on dye and Ca2+ concentrations indicates the presence of both CaAr and CaAr2 complexes, with the CaAr2 form being responsible for the slow, 10-20 ms relaxation of this dye. For antipyrylazo III, the relaxation rate is much faster, less than 1 ms, and the complexing kinetics can be covered with only a CaAp complex. Unlike arsenazo III, antipyrylazo III binding with Ca2+ is rate-limited by a slow structural transition in the dye, taking antipyrylazo III from a low- to a high-affinity structure for Ca2+.

Kinetic scheme for Ca2+-arsenazo III interactions.

Temperature-jump relaxation kinetic studies show that the complex formation between Ca2+ and the metallochromic dye arsenazo III (Ar) is associated with a rapid mode (less than or equal to 10 mus-range) involving both Ca2+ and Na+ of the Na-salt of Ar and a slower mode (approximately 10 ms range) which can be attributed to structural rearrangements in the 1:2 complex CaAr2. The kinetic data suggest the scheme: Ca+2Ar = CaAr+Ar = CaAr2 = CaAr2. The relatively slow rate-limiting step sets a limit for the use of arsenazo III to study the kinetics of Ca2+ processes in cell biology.

Spectrophotometric determination of reaction stoichiometry and equilibrium constants of metallochromic indicators. III. Antipyrylazo III complexing with Ca2+ and acetylcholine receptor protein.

Stoichiometries, equilibrium constants and optical extinction coefficients of calcium-antipyrylazo III (An) complexing are determined with the analytical method described in article I of this series. Spectrophotometric Ca titrations of An at the wavelengths 595 and 710 nm indicate overall dissociation equilibrium constants for the complexes CaAn, CaAn2 and Ca2An to be 4.5 x 10(-4) M, 1.1 x 10(-8) M2 and 1.5 x 10(-6) M2, respectively, extrapolated to zero ionic strength. Ca titrations of solutions containing An plus acetylcholine receptor protein give clear evidence that An binds to the protein to a large extent in the presence of Ca2+; furthermore, addition of acetylcholine results in release of protein-bound Ca and An. This is the first reported indication that antipyrylazo III binds to biological material and questions the usefulness of this dye as a Ca indicator in biological systems.

Spectrophotometric determination of reaction stoichiometry and equilibrium constants of metallochromic indicators. I. General calculational method.

A calculational method is developed for spectrophotometric determination of stoichiometrics and individual equilibrium constants in the complexing of metal ions with metallochromic indicators. Implicit expressions are developed for the calculation of all parameters necessary to describe mixtures of 1 : 1, 1 : 2 and 2 : 1 metal-indicator complexes. The analysis of titration curves entails a series of one-variable best-fit determinations based on mass action and conservation laws; this reduction in the number of degrees of freedom in the curve-fitting procedure yields greater resolution of the complexing parameters than is allowed by conventional methods. Since a common application of metallochromic indicators is to the determination of metal-binding properties of biological molecules, accurate description of metal-indicator complexing is vital for investigation of the regulatory roles of metal ions in biological events.

Spectrophotometric determination of reaction stoichiometry and equilibrium constants of metallochromic indicators. II. The Ca2+-arsenazo III complexes.

The analytical method described in the preceding article was applied to spectrophotometric Ca2+-titrations of the metallochromic indicator arsenazo III (Ar). At various reactant concentrations it was determined that Ar forms 1:1,1:2 and 2 : 1 complexes with calcium. The equilibrium constants and extinction coefficients at 602 nm were determined. Corrected to zero ionic strength at 293 K and pH 7.0, the reactions Ca + Ar = CaAr, CaAr + Ar = CaAr2 and CaAr + Ca = Ca2Ar are associated with dissociation equilibrium constants k(11) = 1.6 x 10(-6)M, K12 = 3.2 x 10(-4)M and K21 = 5.8 x 10(-3)M. respectively. The extinction coefficient of unbound indicator is (602) = 9.6 (+/-0.3) x 10(3) cm(-1) M(-1). Arscnazo III complexes with monovalent ions like Na+ and K+ : at zero ionic strength, the dissociation constant of the Na+-Ar complex is about 0.1 M.

Kinetic models suggest bimolecular reaction steps in axonal Na+-channel gating.

Abstract kinetic models that can successfully simulate the ion-permeability features of axonal Na+ channels suggest the presence of bimolecular reaction steps in the activation of the channels. A chemically plausible interpretation of minimum complexity is described. The implied chemical formalism is highly suggestive of an activator-controlled gating system with strong similarities to the acetylcholine-regulated system. Conformational changes that underlie the ion-conductance changes are suggested to possess a greater sensitivity to the membrane field in axonal parts of excitable membranes than at synaptic parts. This would allow axonal permeability changes to be energetically regulated more conservatively than is observed for synaptic ion channels. Axonal K+ channels with delayed activation kinetics would serve to reverse the increase in membrane permeability to Na+ with a minimum of chemical dissipation.

Theoretical implication of liganding reactions in axonal sodium channel gating.

Mathematical simulation models for measured permeability properties of axonal Na(+) channels point to the presence of bimolecular, as well as net-membrane-field-dependent intramolecular, reaction steps in channel gating reactions. An abstract chemical reaction model is presented, which postulates ligand binding reactions in both Na(+) channel activation and subsequent desensitization as observed in voltage-clamp experiments. Membrane capacitance currents due to the movement of charged or dipolar gating structures also indicate that the early phase of the gating charge movement is dictated by local, rather than net-membrane-field-driven reactions, and therefore reflect energy input from chemical sources which are an integral part of the membrane.

Asymmetric displacement currents in giant axons and macromolecular gating processes.

An electrical-chemical gating model is proposed that describes basic observations on asymmetric displacement currents and transient Na+ conductivity changes in squid giant axons. A previously developed single-parameter analysis of primary voltage clamp data yields normal mode relaxation times that agree well with the time constants of asymmetric capacitative currents, suggesting these currents as gating currents associated with charge displacement in a subunit of a complex gating system. The physical-chemical approach correlates the opening of Na+ channels with charge-charge interactions amongst displaceable membrane charges or dipoles and conformational changes in gating macromolecules. The model covers the close correspondence between the voltage dependence of the peak value of the Na+ conductance change and that of the square of the total displaced charge for small depolarizing voltage steps. The quadratic charge relationship also describes the two-mode relaxation of asymmetric displacement currents; the transiently inhibited return transition of two-thirds of the displaced charge after a prolonged depolarization is interpreted to reflect a dissipative chemical gating process.