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A K Solomon

Publications and source records attributed to A K Solomon.

At least 19 recordsLinked to original sources
PubMed1992

Effect of pCMBS on anion transport in human red cell membranes.

The kinetics of binding of the mercurial sulfhydryl reagent, pCMBS (p-chloromercuribenzene sulfonate), to the extracellular site(s) at which pCMBS inhibits water and urea transport across the human red cell membrane, have previously been characterized. To determine whether pCMBS binding alters Cl- transport, we measured Cl-/NO3- exchange by fluorescence enhancement, using the dye SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium). An essentially instantaneous extracellular phase of pCMBS inhibition is followed by a much slower intracellular phase, correlated with pCMBS permeation. We attribute the instantaneous phase to competitive inhibition of Cl- binding to band 3 by the pCMBS anion. The ID50 of 2.0 +/- 0.1 mM agrees with other organic sulfonates, but is very much greater than that of pCMBS inhibition of urea and water transport, showing that pCMBS reaction with water and urea transport inhibition sites has no effect on anion exchange. The intracellular inhibition by 1 mM pCMBS (1 h) is apparently non-competitive with Ki = 5.5 +/- 6.3 mM, presumably an allosteric effect of pCMBS binding to an intracellular band 3-related sulfhydryl group. After N-ethylmaleimide (NEM) treatment to block these band 3 sulfhydryl groups, there is apparent non-competitive inhibition with Ki = 2.1 +/- 1.2 mM, which suggests that pCMBS reacts with one of the NEM-insensitive sulfhydryl groups on a protein that links band 3 to the cytoskeleton, perhaps ankyrin or bands 4.1 and 4.2.

4-Chloromercuribenzenesulfonate↗
PubMed1991

Transport parameters in the human red cell membrane: solute-membrane interactions of amides and ureas.

We have studied the permeability of a series of hydrophilic amides and ureas through the red cell membrane by determining the three phenomenological coefficients which describe solute-membrane interaction: the hydraulic permeability (Lp), the phenomenological permeability coefficient (omega i) and the reflection coefficient (sigma i). In 55 experiments on nine solutes, we have determined that the reflection coefficient (after a small correction for solute permeation by membrane dissolution) is significantly less than 1.0 (P less than 0.003, t-test), which provides very strong evidence that solute and water fluxes are coupled as they cross the red cell membrane. It is proposed that the aqueous channel is a tripartite assembly, comprising H-bond exchange regions at both faces of the membrane, joined by a narrower sieve-specific region which crosses the lipid. The solutes bind to the H-bond exchange regions to exchange their solvation shell with the H-bonds of the channel; the existence of these regions is confirmed by the finding that the permeation of all the amides and ureas requires binding to well-characterized sites with Km values of 0.1-0.5 M. The sieve-specific regions provide the steric restraints which govern the passage of the solutes according to their size; their existence is shown by the findings that: (1) the reflection coefficient (actually the function [1-corrected sigma i]) is linearly dependent upon the solute molecular diameter; and (2) the permeability coefficient is linearly dependent upon solute molar volume. These several observations, taken together, provide strong arguments which lead to the conclusion that the amides and urea cross the red cell membrane in an aqueous pore.

Amides↗
PubMed1991

Conformational changes in human red cell membrane proteins induced by sugar binding.

We have previously shown that the human red cell glucose transport protein and the anion exchange protein, band 3, are in close enough contact that information can be transmitted from the glucose transport protein to band 3. The present experiments were designed to show whether information could be transferred in the reverse direction, using changes in tryptophan fluorescence to report on the conformation of the glucose transport protein. To see whether tryptophan fluorescence changes could be attributed to the glucose transport protein, we based our experiments on procedures used by Helgerson and Carruthers [Helgerson, A. L., Carruthers, A., (1987) J. Biol. Chem. 262:5464-5475] to displace cytochalasin B (CB), the specific D-glucose transport inhibitor, from its binding site on the inside face of the glucose transport protein, and we showed that these procedures modified tryptophan fluorescence. Addition of 75 mM maltose, a nontransportable disaccharide which also displaces CB, caused a time-dependent biphasic enhancement of tryptophan fluorescence in fresh red cells, which was modulated by the specific anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate). In a study of nine additional disaccharides, we found that both biphasic kinetics and DBDS effects depended upon specific disaccharide conformation, indicating that these two effects could be attributed to a site sensitive to sugar conformation. Long term (800 sec) experiments revealed that maltose binding (+/- DBDS) caused a sustained damped anharmonic oscillation extending over the entire 800 sec observation period. Mathematical analysis of the temperature dependence of these oscillations showed that 2 microM DBDS increased the damping term activation energy, 9.5 +/- 2.8 kcal mol-1 deg-1, by a factor of four to 39.7 +/- 5.1 kcal mol-1 deg-1, providing strong support for the view that signalling between the glucose transport protein and band 3 goes in both directions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

Transport parameters in the human red cell membrane: solute-membrane interactions of hydrophilic alcohols and their effect on permeation.

A systematic study has been made of the three coefficients that describe the human red cell membrane transport of a series of short straight-chain hydrophilic alcohols: the permeability coefficient, omega i, the reflection coefficient, sigma i, and the hydraulic conductivity, Lp. Ethylene glycol transport is saturable with Km = 220 +/- 50 mM; there is a second, low-affinity, ethylene glycol site which inhibits water transport (K = 570 +/- 140 mM, max. inhib. = 90 +/- 10%). sigma eth gly = 0.71 +/- 0.04 which is significantly less than 1 (n = 6, P less than 0.001), as are sigma i for six other alcohols (n = 23), thus providing strong thermodynamic evidence that water and these alcohols cross the red cell membrane, at least in part, in an aqueous channel. The solute/membrane frictional coefficient, fsm, for all seven alcohols has been determined and found to decrease monotonically as membrane permeability increases. The red cell membrane has been perturbed by treatments with phenylglyoxal and BS3 (bis(succinimidyl suberate]; these treatments are accompanied by correlated modulation of both ethylene glycol and urea permeability. In one set of experiments in control cells, urea permeability is correlated with water permeability; and, in another set, ethylene glycol permeability is correlated with water permeability. All of these observations support the proposition that the urea class of solutes, the ethylene glycol class of solutes and water all cross the membrane through the same aqueous pore. A schematic model of the red cell pore, consistent with the experimental observations, is presented.

Alcohols↗
PubMed1990

High proton flux through membranes containing antidiuretic hormone water channels.

Antidiuretic hormone (ADH) stimulation of toad urinary bladder granular cells causes simultaneous increases in transepithelial water and H+ permeabilities (PF and PH+, respectively), suggesting that ADH-elicited water channels inserted into granular cell apical membranes might be permeable to both water and H+. We have previously used self-quenching fluorophores entrapped within endocytic vesicles selectively retrieved from water-permeable apical membranes to measure vesicle PF. The membranes of these vesicles possess an extremely high PF such that our measurements provide only minimum estimates of vesicle PF and have limited our ability to quantitate the properties of ADH water channels. We therefore quantitated vesicle PH+ using similar rapid mixing techniques. Vesicle PH+ was 5.1 +/- 0.5 x 10(-3) cm/s. Activation energy of this process was 3.6 +/- 0.6 kcal/mol, indicative of H+ flux through an aqueous channel. The mercurial reagent, para-chloromercuribenzenesulfonate (PCMBS), which inhibits ADH-stimulated transepithelial PF in intact bladders by 50-60%, inhibited vesicle PH+ by 55%. N-Ethylmaleimide and phloretin, which do not alter ADH-stimulated PF, did not affect vesicle PH+. We conclude that membranes containing ADH water channels possess substantial PH+ that likely reflects proton flux through water channels. The apparent high PH+ of the ADH water channel may have important implications for intracellular trafficking of these water channels in ADH-responsive epithelial cells.

4-Chloromercuribenzenesulfonate↗
PubMed1989

Interaction among anion, cation and glucose transport proteins in the human red cell.

The time course of binding of the fluorescent stilbene anion exchange inhibitor. DBDS (4.4'-dibenzamido-2.2'-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant. tau DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID50 (0.3 +/- 0.1 microM) for H2-DIDS (4.4'-diisothiocyano-2.2'-dihydrostilbene disulfonate) inhibition of tau DBDS is virtually the same as the ID50 (0.47 +/- 0.04 microM) for H2-DIDS inhibition of red cell Cl- flux, thus relating tau DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in tau DBDS, which can be reversed with intracellular, but not extracellular, D-glucose, ID50 for cytochalasin B modulation of tau DBDS is 0.1 +/- 0.2 microM in good agreement with KD = 0.06 +/- 0.005 microM for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 microM), the specific inhibitor of red cell Na+,K+-ATPase, increases red cell Cl- exchange flux in red cells by a factor of about two. This interaction indicates that the Na+,K+-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport protein complex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1989

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells.

In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, tau TDBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID50D,DBDS,MCD (0.5 +/- 0.1 microM) for the H2-DIDS (4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate) inhibition of tau DBDS is in agreement with the ID50,Cl-MCD (0.94 +/- 0.07 microM) for H2-DIDS inhibition of MCD cell Cl- flux, thus relating tau DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl- exchange by a factor of two, from tau Cl- = 0.30 +/- 0.02 sec to 0.56 +/- 0.06 sec (30 mM NaHCO3). The ID50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003 +/- 0.001 microM, so that binding is about an order of magnitude tighter than that for inhibition of rbc K+ flux (KI,K+,rbc = 0.017 microM). These experiments indicate that the Na+,K+-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells. modulates DBDS binding kinetics with a physiological ID50,DBDS,MCD (0.076 +/- 0.005 microM); 2 microM CE also more than doubles the Cl- exchange time constant from 0.20 +/- 0.04 sec to 0.50 +/- 0.08 sec (30 mM NaHCO3). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Is an intact cytoskeleton required for red cell urea and water transport?

In order to determine the membrane protein(s) responsible for urea and water transport across the human red cell membrane, we planned to reconstitute purified membrane proteins into phosphatidylcholine vesicles. In preparatory experiments, we reconstituted a mixture of all of the red cell integral membrane proteins into phosphatidylcholine vesicles, but found that p-chloromercuribenzenesulfonate (pCMBS), which normally inhibits osmotic water permeability by approximately 90%, has no effect on this preparation. The preparation was also unable to transport urea at the high rates found in red cells, though glucose transport was normal. White ghosts, washed free of hemoglobin and resealed, also did not preserve normal urea and pCMBS-inhibitable water transport. One-step ghosts, prepared in Hepes buffer in a single-step procedure, without washing, retained normal urea and pCMBS-inhibitable water transport. Perturbations of the cytoskeleton in one-step ghosts, by removal of tropomyosin, or by severing the ankyrin link which binds band 3 to spectrin, caused the loss of urea and pCMBS-inhibitable water transport. These experiments suggest that an unperturbed cytoskeleton may be required for normal urea and pCMBS-inhibitable water transport. They also show that the pCMBS inhibition of water transport is dissociable from the water transport process and suggest a linkage between the pCMBS water transport inhibition site and the urea transport protein.

4-Chloromercuribenzenesulfonate↗
PubMed1988

Sites of p-chloromercuribenzenesulfonate inhibition of red cell urea and water transport.

The mercurial sulfhydryl reagent, p-chloromercuribenzene sulfonate (pCMBS), inhibits water and urea fluxes across the human red blood cell membrane. The kinetics and affinities for pCMBS binding to separate water transport and urea transport inhibition sites were previously determined by Toon and Solomon ((1986) Biochim. Biophys. Acta 860, 361-375) in red cells that had been treated with N-ethyl-maleimide (NEM) to block five of the six sulfhydryls on the red cell anion exchange protein, band 3. We have used autoradiographs of gels from NEM-treated cells, labeled with 203Hg-pCMBS, to localize these water and urea transport inhibition binding sites separately and find that both are on band 3. Each site is saturable and the time course of each uptake can be fitted to the equation for a bimolecular association (with negligible dissociation) with time constants in agreement with those of Toon and Solomon. Determination of the binding stoichiometry shows one urea inhibition site and three water inhibition sites for every four band 3 molecules. These results indicate that band 3 plays a role in both urea and water transport and suggest that the functional unit may be a tetramer.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1988

Modulation of water transport in human red cells: effect of urea.

We have studied the effect of urea on water flux in the human red cell and have found that 500 mosmolal urea inhibits osmotic water transport by 39%. The Ki for urea inhibition of water flux is 550 +/- 80 mosmolal, higher than, but comparable with, the Km of urea transport into the red cell of 220-330 mM given by Mayrand and Levitt (J. Gen. Physiol. 55 (1983) 427) and Brahm (J. Gen. Physiol. 82 (1983) 1). Other amides, such as propionamide and valeramide, as well as methyl-substituted ureas, have similar effects, although an indifferent molecule, such as 0.5 M creatinine, has no effect. Urea can be washed off the inhibition site with buffer, and the effects of urea concentrations as high as 1.2 osmolal are entirely reversible. 500 mosmolal urea also significantly increases the reflection coefficient for ethylene glycol, sigma eth gly, from 0.71 +/- 0.03 in control experiments to 0.86 +/- 0.04 (P less than 0.0005, t-test), and propionamide has a similar effect on sigma eth gly. These results show that urea can modulate ethylene glycol transport through the red cell membrane, and are consistent with, but not proof of, the presence of a single class of aqueous channels through which both ethylene glycol and urea enter the red cell. It is suggested that the physiological purpose of these low-affinity urea sites is to modulate water flow out of the red cell during passage through the regions of 0.5-0.6 M urea in the kidney.

Amides↗
PubMed1988

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3.

A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682-1688, 1986) found that C1-/HCO-3 exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4'-diisothiocyano-2,2'-disulfonic stilbene), with a K1 similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4'-dibenzamido-2,2'-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (Ks1 = 93 +/- 24 nM) and a lower affinity site (Ks2 = 430 +/- 260 nM), which are closely similar to values for the red cell of 110 +/- 51 nM for the high-affinity site and 980 +/- 200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon, J. Gen. Physiol. 81:421-449, 1983). When Cl- replaces citrate in the buffer, the two sites collapse into a single one with Ks1 = 1500 +/- 400 nM, similar to the single Ks1 = 1200 +/- 200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon, J. Membrane Biol. 89:211-223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 microM-1 are characterized by a fast process, tau = 0.14 +/- 0.03 sec, similar to tau = 0.12 +/- 0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell 'band 3' closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.

Animals↗
PubMed1987

Interrelation of ethylene glycol, urea and water transport in the red cell.

The reflection coefficient, sigma j, which measures the coupling between the jth solute and water transport across a semipermeable membrane, varies between 0 and 1.0. Values of sigma j significantly less than 1.0 provide irreversible thermodynamic proof that there is coupling between the transport of solute and solvent and thus that they share a common pathway. We have developed an improved method for measuring sigma and have used it to determine that sigma ethylene glycol = 0.71 +/- 0.03 and sigma urea = 0.65 +/- 0.03, in agreement with many, but not all, previous determinations. Since both of these values are significantly lower than 1.0, they show that there is a common ethylene glycol/water pathway and a common urea/water pathway. Addition of first one and then two methyl groups to urea increases sigma to 0.89 +/- 0.04 for methylurea and 0.98 +/- 0.4 for 1,3-dimethylurea, consistent with passage through an aqueous pore with a sharp cutoff in the 6-7 A region.

Biological Transport↗
PubMed1987

Modulation of water and urea transport in human red cells: effects of pH and phloretin.

It has previously been shown by Macey and Farmer (Biochim. Biophys. Acta 211:104-106, 1970) that phloretin inhibits urea transport across the human red cell membrane yet has no effect on water transport. Jennings and Solomon (J. Gen. Physiol. 67:381-397, 1976) have shown that there are separate lipid and protein binding sites for phloretin on the red cell membrane. We have now found that urea transport is inhibited by phloretin binding to the lipids with a KI of 25 +/- 8 microM in reasonable agreement with the KD of 54 +/- 5 microM for lipid binding. These experiments show that lipid/protein interactions can alter the conformational state of the urea transport protein. Phloretin binding to the protein site also modulates red cell urea transport, but the modulation is opposed by the specific stilbene anion transport inhibitor, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), suggesting a linkage between the urea transport protein and band 3. Neither the lipid nor the protein phloretin binding site has any significant effect on water transport. Water transport is, however, inhibited by up to 30% in a pH-dependent manner by DIDS binding, which suggests that the DIDS/band 3 complex can modulate water transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Control of red cell urea and water permeability by sulfhydryl reagents.

The binding constant for pCMBS (p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 +/- 30 microM and that for urea transport inhibition to be 0.09 +/- 0.06 microM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10(5) times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1986

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3.

The effect of chloride on 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500 mM) + Na citrate] at constant ionic strength (160 or 600 mM), pH 7.4, 25 degrees C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160 mM ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl] = 10 to 120 mM; at 600 mM ionic strength, the DBDS dissociation constant saturates hyperbolically with half-saturating [Cl] = 450 mM. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600 mM ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: (formula; see text) The equilibrium and rate constants of the model at 600 mM ionic strength are K1 = 0.67 +/- 0.16 microM, k2 = 1.6 +/- 0.7 sec-1, k-2 = 0.17 +/- 0.09 sec-1, K'1 = 6.3 +/- 1.7 microM, k'2 = 9 +/- 4 sec-1 and k'-2 = 7 +/- 3 sec-1. The apparent dissociation constants of chloride from band 3, KCl, are 40 +/- 4 mM (160 mM ionic strength) and 11 +/- 3 mM (600 mM ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1985

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.

4-Chloromercuribenzenesulfonate↗