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A K Solomon

Publications and source records attributed to A K Solomon.

At least 37 records · Page 2Linked to original sources
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

Osmotic properties of human red cells.

When an osmotic pressure gradient is applied to human red cells, the volume changes anomalously, as if there were a significant fraction of "nonosmotic water" which could not serve as solvent for the cell solutes, a finding which has been discussed widely in the literature. In 1968, Gary-Bobo and Solomon (J. Gen. Physiol. 52:825) concluded that the anomalies could not be entirely explained by the colligative properties of hemoglobin (Hb) and proposed that there was an additional concentration dependence of the Hb charge (ZHb). A number of investigators, particularly Freedman and Hoffman (1979, J. Gen. Physiol. 74:157) have been unable to confirm Gary-Bobo and Solomon's experimental evidence for this concentration dependence of ZHb and we now report that we are also unable to repeat the earlier experiments. Nonetheless, there still remains a significant anomaly which amounts to 12.5 +/- 0.8% of the total isosmotic cell water (P much less than 0.0005, t test), even after taking account of the concentration dependence of the Hb osmotic coefficient and all the other known physical chemical constraints, ideal and nonideal. It is suggested that the anomalies at high Hb concentration in shrunken cells may arise from the ionic strength dependence of the Hb osmotic coefficient. In swollen red cells at low ionic strength, solute binding to membrane and intracellular proteins is increased and it is suggested that this factor may account, in part, for the anomalous behavior of these cells.

Adult↗
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

Relation between red cell anion exchange and urea transport.

The new distilbene compound, DCMBT (4,4'-dichloromercuric-2,2,2',2'-bistilbene tetrasulfonic acid) synthesized by Yoon et al. (Biochim. Biophys. Acta 778 (1984) 385-389) was used to study the relation between urea transport and anion exchange in human red cells. DCMBT, which combines properties of both the specific stilbene anion exchange inhibitor, DIDS, and the water and urea transport inhibitor, pCMBS, had previously been shown to inhibit anion transport almost completely and water transport partially. We now report that DCMBT also inhibits urea transport almost completely and that covalent DIDS treatment reverses the inhibition. These observations provide support for the view that a single protein or protein complex modulates the transport of water and urea and the exchange of anions through a common channel.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

Urea reflection coefficient for the human red cell membrane.

The reflection coefficient, sigma, is an irreversible thermodynamic parameter which measures the interaction between solute and solvent in passage across a membrane. The initial estimate of Goldstein and Solomon ((1960) J. Gen. Physiol. 44, 1-17) by the zero-time method gave sigma urea = 0.6 for the human red cell membrane and a more recent measurement by Levitt and Mlekoday ((1983) J. Gen. Physiol. 81, 239-253) using a different method gave sigma urea = 0.95. We have now developed a variant of the zero-time method which gives sigma urea = 0.70 +/- 0.02, which is significantly different from 1.0. There has been controversy as to whether urea permeates the human red cell by the same channel used by water or by a different route. The finding that sigma urea is significantly less than 1.0 (actually less than 0.95) makes it possible to discriminate between these two possibilities since completely independent transfer of urea and water mandates a value of sigma urea = 0.95. Values significantly lower than 0.95 can only be achieved if the transport of the solute, urea, is coupled to that of the solvent, water.

Adult↗
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↗
PubMed1985

n-Alkanols and halothane inhibit red cell anion transport and increase band 3 conformational change rate.

The effects of halothane and n-alkanols on band 3, the anion-exchange protein of the red cell membrane, have been characterized by radioactive sulfate exchange and equilibrium and kinetic binding of a fluorescent anion transport inhibitor, 4,4'-dibenzamido-2,2'-stilbenedisulfonic acid (DBDS), with fluorescence and stopped-flow techniques. Ethanol, butanol, hexanol, heptanol, octanol, and decanol inhibit radioactive sulfate efflux from red blood cells in a dose-dependent manner with an average Hill coefficient of 1.3 +/- 0.1. Over a 10(4)-fold range of buffer concentrations, the calculated membrane alkanol concentrations at which anion transport rates are reduced by 50% are 100-200 mM. At 100-300 mM membrane concentrations, halothane and the n-alkanols increase the apparent rate of DBDS binding to band 3 2-3-fold. Analysis of kinetic and equilibrium DBDS binding data shows that these drugs increase the rate of the DBDS-induced conformational change in the DBDS-band 3 complex. Equilibrium DBDS binding studies reveal differences between the actions of short-chain alkanols (ethanol and butanol) and those of long-chain alkanols (hexanol and longer). Short-chain alkanols reduce the equilibrium affinity of DBDS for band 3, while long-chain alkanols have no effect on equilibrium DBDS binding. The results for halothane and long-chain alkanols suggest a nonspecific, lipid-mediated mechanism of anesthetic action, which may be coupled to protein inactivation by an increase in the rate of protein conformational changes resulting in nonfunctional states. The results for short-chain alkanols indicate that they have the same nonspecific actions as the long-chain alkanols but also have specific effects on the stilbene binding site of band 3.

Alcohols↗
PubMed1985

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.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1984

Specific interaction of the water transport inhibitor, pCMBS, with band 3 in red blood cell membranes.

The human red cell anion transport protein, band 3, contains six pCMBS (p-chloromercuribenzene sulfonate) reactive SH groups, five of which react with N-ethylmaleimide. We have carried out equilibrium binding experiments using N-ethylmaleimide-treated red cell ghosts and found that the sulfhydryl reactive water transport inhibitor, pCMBS, inhibits the binding to band 3 of the specific anion exchange inhibitor DBDS (4,4'-dibenzoamido-2,2'-disulfonic stilbene) in a non-competitive manner. Stopped-flow kinetic studies, in which DBDS is mixed with ghosts in the presence of pCMBS, show that pCMBS slows the DBDS induced conformational change in band 3. A non-competitive reaction scheme has been developed which incorporates the quantitative results of equilibrium and kinetic studies. The pCMBS effect on DBDS binding and kinetics is reversed with 5 mM cysteine suggesting a sulfhydryl bond is involved in pCMBS binding to band 3. These data suggest that pCMBS has a specific binding site on band 3, consistent with the hypothesis that band 3 mediates red cell water transport.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1984

Relation between red cell anion exchange and water transport.

A new distilbene compound, 4',4'-dichloromercuric-2,2,2',2'-bistilbene tetrasulfonic acid (DCMBT), has been synthesized for use in studies of anion and water transport in the human red cell. DCMBT combines features of both the specific stilbene anion transport inhibitor, DIDS, and the mercurial water transport inhibitor, pCMBS. This new compound inhibits anion transport almost completely with a Ki of 15 microM. DCMBT also inhibits water transport by about 15-20% with a Ki of about 8 microM. Treatment of red cells with DIDS inhibits the effect of DCMBT on water transport, suggesting that anion transport and water transport are mediated by the same protein.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Effect of thiourea on PCMBS inhibition of osmotic water transport in human red cells.

The organomercurial reagent p-chloromercuribenzene sulfonate (PCMBS) is an inhibitor of osmotic water permeability in the human red cell membrane. We have found that thiourea, when added along with PCMBS to a red cell suspension, interferes with this inhibition and at high enough concentrations prevents the inhibition from developing altogether. For a 2 mM PCMBS concentration Ki = 3 +/- 1 mM. When thiourea is added at a later time, the PCMBS inhibition, which normally takes about 20 min to develop fully, is halted and remains fixed at the value attained by that time. Thiourea also inhibits the reversal of PCMBS inhibition by a 10 mM concentration of cysteine, the half-time for reversal increasing by more than an order of magnitude when [thiourea] = 50 mM. Possible implications for the nature of the water and urea transport pathways across the red cell membrane are discussed.

4-Chloromercuribenzenesulfonate↗
PubMed1984

Role of membrane proteins and lipids in water diffusion across red cell membranes.

When human red cells are treated with the mercurial sulfhydryl reagent, p-chloromercuribenzene sulfonate, osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104-106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton T1 NMR and obtained a value of 4.0 X 10(-3) cm X s-1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of N-phenyl-1-naphthylamine by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel.

4-Chloromercuribenzenesulfonate↗
PubMed1984

Site of red cell cation leak induced by mercurial sulfhydryl reagents.

It has been suggested that the human red cell anion transport protein, band 3, is the site not only of the cation leak induced in human red cells by treatment with the sulfhydryl reagent pCMBS (p-chloromercuribenzene sulfonate) but is also the site for the inhibition of water flux induced by the same reagent. Our experiments indicate that N-ethylmaleimide, a sulfhydryl reagent that does not inhibit water transport, also does not induce a cation leak. We have found that the profile of inhibition of water transport by mercurial sulfhydryl reagents is closely mirrored by the effect of these same reagents on the induction of the cation leak. In order to determine whether these effects are caused by band 3 we have reconstituted phosphatidylcholine vesicles containing only purified band 3. Control experiments indicate that these band 3 vesicles do not contain (Na+ + K+)-ATPase as measured by ATP dephosphorylation. pCMBS treatment caused a significant increase in the cation leak in this preparation, consistent with the view that the pCMBS-induced cation leak in whole red cells is mediated by band 3.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1983

Anion transport inhibitor binding to band 3 in red blood cell membranes.

The inhibitor of anion exchange 4,4'-dibenzoamido-2,2'-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site.

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