Interaction of phloretin with the anion transport protein of the red blood cell membrane

Phloretin is an inhibitor of anion exchange and glucose and urea transport in human red cells. Equilibrium binding and kinetic studies indicate that phloretin binds to band 3, a major integral protein of the red cell membrane. Equilibrium phloretin binding has been found to be competitive with the binding of the anion transport inhibitor, 4,4′-dibenzamido-2,2′-disulfonic stilbene (DBDS), which binds specifically to band 3. The apparent binding (dissociation) constant of phloretin to red cell ghost band 3 in 28.5 mM citrate buffer, pH 7.4, 25°C, determined from equilibrium binding competition, is $\text{1.8 ± 0.1}\text{μM}$. Stopped-flow kinetic studies show that phloretin decreases the rate of DBDS binding to band 3 in a purely competitive manner, with an apparent phloretin inhibition constant of $\text{1.6 ± 0.4}\text{μM}$. The pH dependence of equilibrium binding studies show that it is the charged, anionic form of phloretin that competes with DBDS binding, with an apparent phloretin inhibition constant of 1.4 μM. The phloretin binding and inhibition constants determined by equilibrium binding, kinetic and pH studies are all similar to the inhibition constant of phloretin for anion exchange. These studies suggest that phloretin inhibits anion exchange in red cells by a specific interaction between phloretin and band 3.     

Interaction of phenylisothiocyanate with human erythrocyte band 3 protein I. Covalent modification and inhibition of phosphate transport

The hydrophobic probe phenylisothiocyanate is utilized for chemical modification of human erythrocyte band 3 protein. The binding of phenylisothiocyanate to this protein is characterized in whole erythrocytes, erythrocyte ghost membranes and in isolated band 3 protein. The label, reactive with nucleophiles in their deprotonated form, is found in all three preparations to be covalently bound to band 3 protein. Under saturation conditions, 4–5 mol phenylisothiocyanate are covalently bound per mol protein (molecular weight 95 000). The described modification effects inhibition of phosphate entry into erythrocytes. 50% inhibition of phosphate transport is obtained following a preincubation of erythrocytes with 0.45 mM phenylisothiocyanate. Both phenylisothiocyanate binding and transport inhibition are saturating processes. The relationship of the two parameters is non-linear.     

Inhibition of anion transport associated with chymotryptic cleavages of red blood cell band 3 protein

Right-side-out vesicles derived from red blood cells treated with chymotrypsin retain specific anion transport function (defined as transport sensitive to the specific inhibitor, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS)), even though the transport protein, band 3, is cleaved into two segments of 60 and 35 kdaltons. In contrast, vesicles derived from alkali-stripped ghosts treated with relatively high concentrations of chymotrypsin retain almost no specific anion function. The loss of function appears to be related to additional cleavages of band 3 protein that occur in treated ghosts, the 60-kdalton segment being reduced first to a 17- and then to a 15-kdalton segment and the 35-kdalton segment being reduced to a 9-kdalton segment plus a carbohydrate containing fragment. The chymotryptic cleavages of band 3 protein of ghosts are preferentially inhibited by high ionic strength, the production of the 9-kdalton segment being somewhat slower than that of the 15-kdalton segment. Vesicles derived from ghosts treated with chymotrypsin at different ionic strengths show a graded reduction in specific anion transport activity, but it was not possible to determine, definitively, which of the additional cleavages was inhibitory. In the light of these data and other information, the functional role of the segments of band 3 is discussed.     

Photoaffinity labeling of procaine-binding sites in normal and sickle cell membranes

A photoaffinity probe, procaine azide, was employed to determine the sites of interaction of procaine in normal and sickle cell erythrocytes. Studies show that the number of binding sites and affinity of procaine to membranes derived from normal and sickled cell erythrocytes were similar, although procaine retards the in vitro formation of irreversibly sickled cells from cells. The results show that procaine azide, a photoaffinity analogue of procaine, is covalently incorporated into both protein (60–70%) and lipid (40–30%) components of the membrane. Sodium dodecyl sulfate-gel electrophoresis of the labeled ghosts show that procaine binds specifically to band 3 and periodic acid-Schiff staining bands in membranes derived from labeled erythrocytes. Binding of procaine or covalent incorporation of procaine azide into membrane proteins does not affect the phosphate transport. Moreover, pre-treatment of intact erythrocytes with 4,4′-diisothiocyano-2,2′-stilbene disulfonate, an anion transport inhibitor, did not affect either the binding or covalent incorporation of procaine azide into erythrocytes. These results indicate that the binding of procaine azide to Band 3 protein occurs at a locus different than that involved in anion translocation process.     

The location of a disulfonic stilbene binding site in band 3, the anion transport protein of the red blood cell membrane

The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, a specific, potent, irreversible inhibitor of anion transport in red blood cells is located in a 15 000 dalton transmembrane segment of band 3, produced by chymotrypsin treatment of ghosts stripped of extrinsic proteins. The segment was cleaved into three fragments of 7000, 4000 and 4000 daltons by CNBr. The C-terminus of the segment is located in the 7000 dalton fragment; the N-terminus in one of the 4000 dalton fragments; and the binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid in the middle 4000 dalton fragment. The latter was cleaved by $\text{N-}\text{bromosuccinimide}$ into two fragments of 2000 daltons. The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid was located on the fragment containing the newly formed N-terminus. It is concluded that the binding site is located about 9000 daltons from the C-terminus (at the outside face of the membrane) and 6000 daltons from the N-terminus (at the cytoplasmic face). In view of the existing evidence that the binding site may be located near the outside face of the membrane, it is suggested that the 15 000 dalton segment is folded, so that it crosses the bilayer three times.     

KCl loss and cell shrinkage in the ehrlich ascites tumor cell induced by hypotonic media, 2-deoxyglucose and propranolol

Ehrlich ascites tumor cells lose KCl and shrink after swelling in hypotonic media and in response to the addition of 2-deoxyglucose, propranolol, or the Ca²⁺ ionophore, A23187, plus Ca²⁺ in isotonic media. All of these treatments activate cell shrinkage via a pathway with the following characteristics: (1) the KCl loss responsible for cell shrinkage does not alter the membrane potential; (2) NO₃^? does not substitute for Cl^?; (3) the net KCl movements are not inhibited by quinine or DIDS; and (4) early in this study furosemide was effective in inhibiting cell shrinkage but this sensitivity was subsequently lost. This evidence suggests that the KCl loss in these cells occurs via a cotransport mechanism. In addition, hypotonic media and the other agents used here stimulate a Cl^? -Cl^? exchange, a net loss of K⁺ and a net gain of Na⁺ which are not responsible for cell shrinkage. The Ehrlich cell also appears to have a Ca²⁺-activated, quinine-sensitive K⁺ conductive pathway but this pathway is not part of the mechanism by which these cells regulate their volume following swelling or shrink in isotonic media in response to 2-deoxyglucose or propranolol. Shrinkage by the loss of K⁺ through the Ca²⁺ stimulated pathway appears to be limited by Cl^? conductive movements; for when NO₃^?, an anion demonstrated here to have a higher conductive movement than Cl^?, is substituted for Cl^?, the cells will shrink when the Ca²⁺-stimulated K⁺ pathway is activated.     

Proteolytic cleavages of cytochalasin B binding components of Band 4.5 proteins of the human red blood cell membrane

The putative hexose transport component of Band 4.5 protein of the human erythrocyte membrane was covalently photolabelled with [³H]cytochalasin B. Its transmembrane topology was investigated by electrophoretically monitoring the effect of proteinases applied to intact erythrocytes, unsealed ghosts, and a reconstituted system. Band 4.5 was resistant to proteolytic digestion at the extracellular face of the membrane in intact cells at both high and low ionic strengths. Proteolysis at the cytoplasmic face of the membrane in ghosts or reconstituted vesicles resulted in cleave of the transporter into two membrane-bound fragments, a peptide of about 30 kDa that contained its carbohydrate moiety, and a 20 000 kDa nonglycosylated peptide that bore the cytochalasin B label. Because it is produced by a cleavage at the cytoplasmic face and because the carbohydrate moiety is known to be exposed to the outside, the larger fragment must cross the bilayer. It has been reported that the Band 4.5 sugar transporter may be derived from Band 3 peptides by endogenous proteolysis, but the cleavage pattern found in the present study differs markedly from that previously reported for Band 3. Minimization of endogenous proteolysis by use of fresh cells, proteinase inhibitors, immediate use of ghosts and omission of the alkaline wash resulted in no change in the incorporation of [³H]cytochalasin B into Band 4.5, and no labelling of Band 3 polypeptides. These results suggest that the cytochalasin B binding component of Band 4.5 is not the product of proteolytic degradation of a Band 3 component.     

Formation of aqueous pores in the human erythrocyte membrane after oxidative cross-linking of spectrin by diamide

Oxidation of erythrocyte membrane SH-groups by diamide and tetrathionate induces cross-linking of spectrin (Haest, C.W.M., Kamp, D., Plasa, G. and Deuticke, B. (1977) Biochim. Biophys. Acta 469, 226–230). This cross-linking was now shown to go along with a concentration- and time-dependent enhancement of membrane permeability for hydrophilic nonelectrolytes and ions. The enhancement is specific for oxidative SH-group modifications, is reversible by reduction of the induced disulfides, can be suppressed by a very brief pre-treatment of the cells with low concentrations of $\text{N-}\text{ethylmaleimide}$ and is strongly temperature-dependent. The pathway of the induced permeability discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy ($\text{E}{}_{\mathrm{<mtext>a</mtext>}}\text{3–8}\text{kcal/mol}$). These findings are reconcilable with the formation of a somewhat inhomogeneous population of aqueous pores with radii probably $\text{? 0.65}\text{nm}$. Estimated pore numbers vary with the size of the probe molecule. Assuming a diffusion coefficient as in bulk water within the pore, at least 20 pores per cell have to be postulated; more realistic lower diffusion coefficients increase that number. Alterations of the lipid domain by changes of cholesterol contents and insertion of hexanol or nonionic detergents alter the number or size of the pores. Since aggregation of skeletal and intrinsic membrane proteins also occurs after the SH-oxidation, in parallel to the formation of membrane leaks, one may consider (a) defects in the disturbed bilayer interface, (b) a mismatch between lipid and intrinsic proteins or (c) channels inbetween aggregated intrinsic proteins as structures forming the pores induced by diamide treatment.     

Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane

The hemoglobin binding sites on the inner surface of the erythrocyte membrane were identified by measuring the fraction of hemoglobin released following selective proteolytic or lipolytic enzyme digestion. In addition, binding stoichiometry to and fractional hemoglobin release from inside-out vesicle preparations of human and rabbit membranes were compared since rabbit membranes differ significantly from human membranes only in that they lack glycophorin. Our results show that rabbit inside-out vesicles bind about 65% less human or rabbit hemoglobin under conditions of optimal and stoichiometric binding, despite being otherwise similar in composition. We suggest that this difference is either directly or indirectly due to the absence of glycophorin in rabbit membranes. Further supportive evidence includes demonstrating (a) that neuraminidase treatment of human membranes did not affect hemoglobin binding and (b) that reconstitution of isolated glycophorin into phospholipid vesicles increased the hemoglobin binding capacity in a manner proportional to the fraction of glycophorin molecules oriented with their cytoplasmic sides exposed to the exterior of the vesicle. Proteolysis of human inside-out vesicles either before or after addition of hemoglobin reduced the binding capacity by about 25%. This is consistent with the known proportion of total hemoglobin binding sites involving band 3 protein and the selective lability of the cytoplasmic aspect of band 3 protein to proteolysis. Phospholipid involvement in hemoglobin binding was determined using various phospholipase C preparations which differ in their reactivity profiles. Approximately 38% of the bound hemoglobin was released upon cleavage of phospholipid headgroups. These results suggest that the predominant sites of binding for hemoglobin on the inner surface of the red cell membrane are the two major integral membrane glycoproteins.     

The locations of the three cysteine residues in the primary structure of the intrinsic segments of band 3 protein,and implications concerning the arrangement of band 3 protein in the bilayer

The intrinsic domains of band 3 protein contain three cysteine residues, one in a 17 kDa middle segment and two in a 35 kDa C-terminal segment. The latter are retained in an 8 kDa fragment produced by chymotrypsin treatment of ghosts. Cleavage of cysteine residues by 2-nitro-5-thiocyanobenzoic acid (NTCB) allows localization of this amino acid in the primary structure of the 8, 17, 35 and 52 (17 plus 35) kDa segments of band 3 protein. The mapping of these residues taken with other information concerning accessibility of various sites at the two sides of the membrane leads to the conclusion that band 3 protein crosses the membrane at least five times, or ten times in a dimer structure. The implications of this conclusion in terms of band 3 protein structure and function are briefly discussed.     

Fluorescence labeling of the human erythrocyte anion transport system. Subunit structure studied with energy transfer

The anion transport system of human red cells was isolated in vesicles containing the original membrane lipids and the 95 000 dalton polypeptides (band 3) by the method of Wolosin et al. (J. Biol. Chem. (1977) 252, 2419–2427). The vesicles have a functional anion transport system since they display sulfate transport that is inhibited by the fluorescent probe 8-anilinonaphthalene 1-sulfonate (ANS) with similar potency as in red cells. The vesicles were labeled with the SH-specific probe fluorescein mercuric acetate (FMA). Labeling lowers FMA fluorescence, and is prevented or reversed by dithiothreitol, suggesting that the reaction is with a thiol group on the protein. Fluorescence titrations show a maximum labeling stoichiometry of 1.3 ± 0.4 mol FMA/mol 95 000 dalton polypeptide. The polarization of bound FMA fluorescence is high indicating that the probe is highly immobilized. Pretreatment with Cu²⁺ + o-phenanthroline under conditions that crosslink band 3 in ghosts decreases FMA labeling 50%. Differences in kinetics of FMA labeling in sealed and leaky vesicles suggest that the reactive SH group is located in the intravesicular portion of the protein (corresponding to the cytoplasmic surface of the red cell) and that FMA can cross the membrane. Inhibitors of anion transport have no effect on FMA labeling kinetics suggesting it is not transported via the anion     

The amino acid conjugate formed by the interaction of the anion transport inhibitor 4,4′-diisothiocy ano-2,2′- stilbenedisulfonic acid (DIDS) with band 3 protein from human red blood cell membranes

The specific anion transport inhibitor 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and its reduced analog (H₂DIDS), when irreversibly bound to band 3 protein of the red blood cell membrane, form amino acid conjugates through interaction with the ?-amino group of a particular lysine residue. The specific residue is located in a transmembrane segment of band 3 protein and appears to be a close neighbor of the transport site.     

Inhibition of α-aminoisobutyric acid uptake by diisothiocyanostilbenedisulphonic acid in the amphibian cornea

α-Aminoisobutyric acid accumulation of the toad's (Bufo marinus) cornea and lens is inhibited by 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid. This effect is seen at pH 8.4; at pH 7.4 a small increase in aminoisobutyric acid uptake was observed. Efflux of aminoisobutyric acid is unchanged by diisothiocyanostilbenedisulphonic acid at either pH. The inhibitory effect of diisothiocyanostilbenedisulphonic acid on aminoisobutyric acid accumulation appears to reflect a direct action on membrane mechanisms that mediate its influx.     

Small-intestinal Na+/d-glucose cotransport. Inactivation of sugar transport and phlorizin binding by thiol-group and amino-group reagents

It has previously been shown that mercurials acting from the cytoplasmic side or from within the hydrophobic part of the membrane inactivate the small intestinal Na⁺/d-glucose cotransporter by blocking essential SH-groups (Klip, A., Grinstein, S. and Semenza, G. (1979) Biochim. Biophys. Acta 558, 233–245). Another (set of) sulfhydryl(s) which are critical for phlorizin binding and sugar transport function and which may lie on the luminal side of the brush border membrane, can be blocked by DTNB and 4,4′-dithiopyridine but not by $\text{N-}\text{ethylmaleimide}$. In addition, modification of amino groups by fluorescamine, reductive methylation and (under certain conditions) DIDS also lead to inactivation of the carrier's binding and transport functions. No evidence was obtained that any of the above groups is directly involved in the binding of either Na⁺/d-glucose or phlorizin, since none of these compounds prevented inactivation of the cotransporter.     

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 K_i = ; 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.     

Phospholipid dependence of the anion transport system of the human erythrocyte membrane. Studies on reconstituted band 3/lipid vesicles

Band 3 protein extracted from human erythrocyte membranes by Triton X-100 was recombined with the major classes of phospholipid occurring in the erythrocyte membrane. The resulting vesicle systems were characterized with respect to recoveries, phospholipid composition, protein content and vesicle size as well as capacity and activation energy of sulfate transport. Transport was classified into band-3-specific fluxes and unspecific permeability by inhibitors. Transport numbers (sulfate ions per band 3 per minute) served as a measure of functional recovery after reconstitution. The transport properties of band 3 proved to be insensitive to replacement of phosphatidylcholine by phosphatidylethanolamine, while sphingomyelin and phosphatidylserine gradually inactivated band-3-specific anion transport when present at mole fractions exceeding 30 mol%. The activation energy of transport remained unaltered in spite of the decrease in transport numbers. The results, which are discussed in terms of requirements of band 3 protein function with respect to the fluidity and surface charge of its lipid environment, provide a new piece of evidence that the transport function of band 3 protein depends on the properties of its lipid environment just as the catalytic properties of some other membrane enzymes. The well-established species differences in anion transport (Gruber, W. and Deuticke, B. (1973) J. Membrane Biol. 13, 19–36) may to some extent reflect this lipid dependence.     

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ($\text{I}{}_{50}\text{= 6·10}{}^{\mathrm{?7}}\text{M}$). The concentration dependence of the binding to ghosts reveals two saturable components. [¹⁴C]Flufenamate binds with high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_1\text{= 1.2·10}{}^{\mathrm{?7}}\text{M}$) to 8.5·10⁵ sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_2\text{= 10}{}^{\mathrm{?4}}\text{M}$) to a second set of sites (4.6·10⁷ per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [¹⁴C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [¹⁴C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [¹⁴C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [¹⁴C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.     

Discrimination of three parallel pathways of lactate transport in the human erythrocyte membrane by inhibitors and kinetic properties

The transmembrane movements of lactate and other monocarboxylate anions in mammalian erythrocytes have been claimed, by virtue of their sensitivity to SH-reagents, to involve a transfer system different from the classical anion system (Deuticke, B., Rickert, I. and Beyer, E. (1978) Biochim. Biophys. Acta 507, 137–155). Inhibition of monocarboxylate transfer by SH-reagents, however, was incomplete to an extent varying for different monocarboxylates. The transport component insensitive to SH-reagents has now been shown to involve (a) the classical anion-exchange system, as demonstrated by sensitivity to specific disulfonate inhibitors, and (b) nonionic diffusion, as indicated by the characteristic pH- and concentration dependency of this component and its stimulation by aliphatic alcohols. Under physiological conditions about 90% of total lactate movement proceed via the specific system, 5% via the classical anion-transfer system, 5% by nonionic diffusion. These three components of lactate exchange differ in their activation energies. The specific lactate system mediates net fluxes almost as fast as exchange fluxes, in marked contrast to the classical anion-exchange system which mediates halide exchange much faster than halide net movements. The underlying mechanism, for maintenance of electroneutrality, is an OH^?-antiport or an H⁺-symport as indicated by the particular response of lactate net fluxes to changes of intra- or extracellular pH.     

Effect of staphylococcal alpha-hemolysin upon anion transport in the rabbit erythrocyte

Equilibrium exchange of SO₄^2? was measured prior to and during hemolysis in rabbit erythrocytes exposed to staphylococcal α-hemolysin. The anion-transport protein of the rabbit erythrocyte has also been identified. Equilibrium exchange of SO₄^2? was measured by both efflux and influx of ³⁵SO₄^2?. The rate of influx of SO₄^2? in rabbit erythrocytes exposed to α-hemolysin was twice that of the untreated cells. The rate of SO₄^2? efflux was unchanged by α-hemolysin. Inhibition of anion exchange with 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) did not inhibit hemolysis, therefore, the increased influx of SO₄^2? may occur through a DIDS-insensitive pathway.     

Partial characterization of the glucose transport activity in the golgi-rich fraction of fat cells

The glucose transport activity solubilized from the basal and plus insulin forms of the Golgi-rich fraction of adipocytes was partially characterized, and the results were compared with those of the activity obtained from the plus insulin form of the plasma membrane-rich fraction. The transport activity was determined in a cell-free, reconstituted, system. Prior to reconstitution, the activities in the three preparations were all (a) stable at 0°C for at least 4 h, but not at 37°C or above; (b) most stable at pH 7–9, and (c) less stable in Tes than in Tris buffer. After reconstitution, the three activities were all (d) stable at 0°C, (e) most active at pH 5.5, (f) mildly stimulated by divalent cations, (g) unaffected by insulin or 1 mM of several SH-blocking agents, (h) inhibited by heavy metal ions, 10–100 mM of monovalent salts, organic solvents, several sugar isomers, and specific sugar-transport inhibitors. The rates of d-glucose uptake by the three liposome preparations were all inhibited more strongly by 2-deoxy-d-glucose or $\text{3}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ than by d-glucose. These data indicate that the general properties of the glucose transport activity in the Golgi-rich fraction are similar to those of the activity in the plasma membrane-rich fraction.