Resolution and reconstitution of anion exchange reactions

To illustrate the emerging class of anion exchange proteins in bacteria, this article discusses the biochemical and physiological properties of phosphate (Pi)-linked antiporters that accept glucose 6-phosphate (G6P) as their primary substrate. These systems have a bifunctional active site that binds a pair of negative charges, whether presented as a single divalent anion or a pair of monovalent substrates. Exchange stoichiometry therefore moves between the limits of 2:1 and 2:2 according to the ratio of mono- and divalent substrates at either membrane surface. This predicts an interesting reaction sequence in vivo because internal pH is more alkaline than external pH; one expects an asymmetric exchange as a pair of monovalent G6P anions moves against a single divalent G6P, and in this way an otherwise futile self-exchange of G6P can result in a net inward flux driven (indirectly) by the pH gradient. Despite their biochemical complexity, at a molecular level the Pi-linked antiporters resemble other secondary carriers. Indeed, the current listing of nearly two dozen such proteins suggests a structural theme in which the minimal functional unit has two sets of six transmembrane alpha helices separated by a central hydrophilic loop. Presently described examples show that this topology can derive from either a single protein or from pairs of identical subunits. The finding of this common structure makes it possible to begin building more detailed structural models that have more general implications.     

Anion exchange reactions in bacteria

Bacterial anion exchange now includes both carboxylate-linked reactions, in which there is an antiport of mono- and dicarboxylic acids, and Pi-linked reactions that build on phosphate (Pi) and organic phosphates. To illustrate the general features of this expanding class, this article discussed the biochemistry, physiology, and molecular biology of Pi-linked antiporters that accept glucose 6-phosphate (G6P) as their primary substrate. Kinetic and biochemical analysis suggsts that Pi-linked exchangers have a bifunctional active site that accepts a pair of negative charges. For this reason, exchange stoichiometry moves between the limits of 2:1 and 2:2 to reflect the ratio of mono- and divalent substrates at either membrane surface. This results in a particularly interesting reaction sequencein vivo, where, because cytosolic pH is relatively alkaline, one can expect the asymmetric exchange of two monovalent G6P anions against a single divalent G6P. In this way, an otherwise futile self-exchange of G6P gives a net flux driven (indirectly) by the pH gradient. Despite this biochemical and physiological complexity, Pi-linked carriers resemble all other secondary carriers at a molecular level. Indeed, sequence analysis leads one to infer a common (albeit low resolution) structural theme in which each functional unit has two sets of six trans-membrane helices separated by a central hydrophilic loop. Present examples show that this topology can derive from either a single protein, as is typical in bacteria, or from pairs of identical subunits, as found in mitochondria and chloroplasts. The finding of this common structure should make it possible to build detailed structural models that have implications for all membrane carrier proteins.     

Identification and functional reconstitution of phosphate: sugar phosphate antiport of Staphylococcus aureus

Resting cells of Staphylococcus aureus displayed a phosphate (Pi) exchange that was induced by growth with glucose 6-phosphate (G6P) or sn-glycerol 3-phosphate (G3P). Pi-loaded membrane vesicles from these cells accumulated 32Pi, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of 32Pi (and L-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (DL-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both Pi and DL-lactate to establish an internal pool of Pi. This trans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal Pi. Reconstitution of membrane protein allowed a quantitative analysis of Pi-linked exchange. Pi-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous 32Pi: Pi exchange (Kt's of 2.2 and 1.4 mM; Vmax's of 180 and 83 nmol Pi/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (Kt = 27 microM) over G3P (Kt = 1.3 mM) and Pi (Kt = 2.2 mM), suggesting that the same antiporter was induced in both cases. We conclude that 32Pi: Pi exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of phosphate:hexose 6-phosphate antiport in membrane vesicles of Streptococcus lactis

Membrane vesicles of Streptococcus lactis were used to characterize a novel anion exchange involving phosphate and sugar 6-phosphates. For vesicles loaded with 50 mM phosphate at pH 7, homologous phosphate:phosphate exchange had a maximal rate of 130 nmol/min/mg of protein and a Kt of 0.21 mM external phosphate; among phosphate analogues tested, only arsenate replaced phosphate. Heterologous exchange was studied by 2-deoxyglucose 6-phosphate entry into phosphate-loaded vesicles; this reaction had a maximal velocity of 31 nmol/min/mg of protein and a Kt of 26 microM external substrate. Sugar phosphate moved intact during this exchange, since its entry led to loss of internal 32Pi without transfer of 32P to sugar phosphate. Inhibitions of phosphate exchange suggested that the preferred sugar phosphate substrates were (Kiapp): glucose, 2-deoxyglucose, and mannose 6-phosphates (approximately 20 microM) greater than fructose 6-phosphate (150 microM) greater than glucosamine 6-phosphate (420 microM) greater than alpha-methylglucoside 6-phosphate (740 microM). Stoichiometry for phosphate:2-deoxyglucose 6-phosphate antiport was 2:1 at pH 7, and since initial rates of exchange were unaffected by charge carrying ionophores (gramicidin, valinomycin, a protonophore), this unequal stoichiometry indicated the electroneutral exchange of two monovalent phosphates for a single divalent sugar phosphate.     

Reconstitution of the phosphoglycerate transport protein of Salmonella typhimurium

Operation of the phosphoglycerate transport protein (PgtP) of Salmonella typhimurium has been studied in proteoliposomes by using a technique in which membrane protein is solubilized and reconstituted directly from small volumes of cell cultures. When protein from induced cells was reconstituted into phosphate (Pi)-loaded proteoliposomes, it was possible to demonstrate a PgtP-mediated exchange of internal and external phosphate. For this homologous Pi:Pi antiport, kinetic analysis indicated a Michaelis constant (Kt) of 1 mM and a maximal velocity of 26 nmol/min mg of protein; arsenate inhibited with a Ki of 1.3 mM, suggesting that PgtP did not discriminate between these two inorganic substrates. Pi-loaded proteoliposomes also accumulated 3-phosphoglycerate and phosphoenolpyruvate, establishing for each of them a concentration gradient (in/out) of about 100-fold; phosphoenolpyruvate (Ki = 70 microM) rather than 3-phosphoglycerate (Kt = 700, Ki = 900 microM) was the preferred substrate for these conditions. We also concluded that such heterologous exchange was a neutral event, since its rate and extent were unaffected by the presence of a protonophore and unresponsive to the imposition of a membrane potential (positive or negative inside). In quantitative work, we found a stoichiometry of 1:1 for the exchange of Pi and 3-phosphoglycerate, and given an electroneutral exchange, this finding is most easily understood as the overall exchange of divalent Pi against divalent phosphoglycerate. These experiments establish that PgtP functions as an anion exchange protein and that it shares important mechanistic features with the Pi-linked antiporters, GlpT and UhpT, responsible for transport of glycerol 3-phosphate and hexose 6-phosphates into Escherichia coli.     

Bacterial anion exchange. Use of osmolytes during solubilization and reconstitution of phosphate-linked antiport from Streptococcus lactis

Membranes of Streptococcus lactis were solubilized with 1.1% octyl-beta-D-glucopyranoside in the presence of 0.37% acetone/ether-washed phospholipid from several sources. After adding excess Escherichia coli phospholipid as bath-sonicated liposomes, phosphate:sugar phosphate antiport was reconstituted in proteoliposomes by a 25-fold dilution in 0.1 M KPi (pH 7). Assays of 32Pi:Pi exchange showed that antiport was subject to an inactivation which varied in severity according to the lipid present at solubilization. Recovery of Pi-linked exchange was improved by the presence of 10-20% glycerol or other osmolyte during extraction. The osmolytes tested in this regard have included polyols (glycerol, erythritol, xylitol, sorbitol), sugars (glucose, trehalose), and two amino acids (glycine, proline). Each gave 10--20-fold increased recoveries of 32Pi:Pi antiport compared to controls using only detergent and lipid; these precautions were not required for the efficient reconstitution of F0F1-ATPase. Antiport in the artificial system was studied most carefully when glycerol was the stabilizing additive. For that case, the Kt values for Pi or 2-deoxyglucose 6-phosphate transport (275 and 25 microM, respectively) were the same as in native membranes. Maximal rates of Pi and 2-deoxyglucose 6-phosphate transport (200 and 42 nmol/min/mg of protein, respectively) and the turnover number for Pi exchange (25--50/s) suggested that antiporters were recovered without loss of activity. We conclude that the quantitative aspects of bacterial anion exchange are amenable to study in an artificial system, and that the use of osmolytes as general stabilants can be a valuable adjunct to current techniques for reconstitution of integral membrane transport proteins.     

pH dependence of phosphate transport across the red blood cell membrane after modification by dansyl chloride

Dansylation of the red blood cell membrane inhibits monovalent anion transport as measured by means of 36C1 and enhances divalent anion transport as measured by means of 35SO4 (Legrum, Fasold and Passow (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1573-1590 and Lepke and Passow (1982) J. Physiol. (London) 328, 27-48). In the present work the effect of dansylation on phosphate equilibrium exchange was studied over the pH range where the ratio between monovalent and divalent phosphate anions varies. At high pH, phosphate equilibrium exchange was enhanced; at low pH, exchange was inhibited. The pH maximum of phosphate equilibrium exchange, seen at pH 6.3 in untreated ghosts is now replaced by a plateau. The inverse effects of dansylation on the rates of exchange at high and low pH suggest that both monovalent and divalent phosphate anions are accepted as substrates by the anion transport protein. A tentative attempt to obtain a quantitative estimate of the ratio of monovalent and divalent phosphate transport indicates that in the untreated red cell membrane over the pH range 7.2-8.5 the transport of HPO42- is negligible compared to the transport of H2PO4-.     

Reconstitution of sugar phosphate transport systems of Escherichia coli

Studies with Escherichia coli cells showed that the transport systems encoded by glpT (sn-glycerol 3-phosphate transport) and uhpT (hexose phosphate transport) catalyze a reversible 32Pi:Pi exchange. This reaction could be used to monitor the glpT or uhpT activities during reconstitution. Membranes from suitably constructed strains were extracted with octylglucoside in the presence of lipid and glycerol, and proteoliposomes were formed by dilution in 0.1 M KPi (pH 7). Both reconstituted systems mediated a 32Pi:Pi exchange which was blocked by the appropriate heterologous substrate, sn-glycerol 3-phosphate (G3P) or 2-deoxyglucose 6-phosphate (2DG6P), with an apparent Ki near 50 microM. In the absence of an imposed cation-motive gradient, Pi-loaded proteoliposomes also transported the expected physiological substrate; Michaelis constants for the transport of G3P or 2DG6P were near 20 microM. The heterologous exchange showed a maximal velocity of 130 nmol/min/mg protein via the glpT system and 11 nmol/min/mg protein for the uhpT system. This difference was expected because the G3P transport activity had been reconstituted from a strain carrying multiple copies of the glpT gene. Taken together, these results suggest that anion exchange may be the molecular basis for transport by the glpT and uhpT proteins.     

Altered substrate selectivity in a mutant of an intrahelical salt bridge in UhpT, the sugar phosphate carrier of Escherichia coli

Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the sugar phosphate carrier of Escherichia coli. Glucose 6-phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp388 or Lys391 but not if both are replaced. This suggests that Asp388 and Lys391 are involved in an intrahelical salt bridge and that neither is required for normal UhpT function. This interpretation is strengthened by the finding that mutations at Lys391 (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant. Further work shows that although the D388C variant is null for G6P transport, movement of 32Pi by homologous Pi/Pi exchange is unaffected. This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the Pi/Pi exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, Pi/Pi exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related Pi-linked antiporter, which lacks the Asp388-Lys391 salt bridge but has instead an uncompensated arginine at position 391. For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP.     

VALENCY RULE AND ALLEGED HOFMEISTER SERIES IN THE COLLOIDAL BEHAVIOR OF PROTEINS : I. THE ACTION OF ACIDS.

1. The action of a number of acids on four properties of gelatin (membrane potentials, osmotic pressure, swelling, and viscosity) was studied. The acids used can be divided into three groups; first, monobasic acids (HCl, HBr, HI, HNO₃, acetic, propionic, and lactic acids); second, strong dibasic acids (H₂SO₄ and sulfosalicylic acid) which dissociate as dibasic acids in the range of pH between 4.7 and 2.5; and third, weak dibasic and tribasic acids (succinic, tartaric, citric) which dissociate as monobasic acids at pH 3.0 or below and dissociate increasingly as dibasic acids, according to their strength, with pH increasing above 3.0. 2. If the influence of these acids on the four above mentioned properties of gelatin is plotted as ordinates over the pH of the gelatin solution or gelatin gel as abscissæ, it is found that all the acids have the same effect where the anion is monovalent; this is true for the seven monobasic acids at all pH and for the weak dibasic and tribasic acids at pH below 3.0. The two strong dibasic acids (the anion of which is divalent in the whole range of pH of these experiments) have a much smaller effect than the acids with monovalent anion. The weak dibasic and tribasic acids act, at pH above 3.0, like acids the anion of which is chiefly monovalent but which contain also divalent anions increasing with pH and with the strength of the acid. 3. These experiments prove that only the valency but not the other properties of the anion of an acid influences the four properties of gelatin mentioned, thus absolutely contradicting the Hofmeister anion series in this case which were due to the failure of the earlier experimenters to measure properly the pH of their protein solutions or gels and to compare the effects of acids at the same pH of the protein solution or protein gel after equilibrium was established. 4. It is shown that the validity of the valency rule and the non-validity of the Hofmeister anion series for the four properties of proteins mentioned are consequences of the fact that the influence of acids on the membrane potentials, osmotic pressure, swelling, and viscosity of gelatin is due to the Donnan equilibrium between protein solutions or gels and the surrounding aqueous solution. This equilibrium depends only on the valency but not on any other property of the anion of an acid. 5. That the valency rule is determined by the Donnan equilibrium is strikingly illustrated by the ratio of the membrane potentials for divalent and monovalent anions of acids. Loeb has shown that the Donnan equilibrium demands that this ratio should be 0.66 and the actual measurements agree with this postulate of the theory within the limits of accuracy of the measurements. 6. The valency rule can be expected to hold for only such properties of proteins as depend upon the Donnan equilibrium. Properties of proteins not depending on the Donnan equilibrium may be affected not only by the valency but also by the chemical nature of the anion of an acid.     

Activation of angiotensin converting enzyme by monovalent anions

The angiotensin converting enzyme catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions in the order C1- greater than Br- greater than F- greater than NO3- greater than CH3COO-. In the alkaline pH region, increasing anion concentrations decrease the KM but do not change the kcat. This behavior is characteristic of an ordered bireactant mechanism in which the anion binds to the enzyme prior to the substrate. At acidic pH values, however, the anion activation is a result of both a decrease in KM and an increase in kcat, implying a bireactant mechanism in which anion and substrate bind randomly. For both the ordered and the bireactant mechanisms the anion serves as an essential activator. The effect of chloride on enzyme activity was studied over the pH range 5-10 under kcat/KM conditions and demonstrates that the apparent chloride binding constant increases from 3.3 mM at pH 6.0 to 190 mM at pH 9.0. The kcat vs. pH profile exhibits two pK values of 5.6 and 9.6, while the variation of KM with pH is characterized by a pK of 8.9 and a 2-fold increase between pH 6.5 and 7.5. The chloride activation of the hydrolysis of furanacryloyl-Phe-Gly-Gly is compared with that of the physiological substrates angiotensin I and bradykinin.     

Monoanion inhibition and 35Cl nuclear magnetic resonance studies of renal dipeptidase.

Kinetic analyses of monoanion inhibition and 15Cl nuclear magnetic resonance at 5.88 MHz were employed to study monoanion interactions with the zinc metalloenzyme, renal dipeptidase. The enzyme-catalyzed hydrolysis of glycyldehydrophenylalanine exhibited competitive inhibition when the reaction rate was determined in the presence of the monovalent anions fluoride, chloride, bromide, iodide, azide, nitrate, or thiocyanate or upon the addition of the divalent anion, sulfate. Competitive inhibition was produced by these anions. One anion was bound per enzyme molecule, and except in the case of fluoride all of the anions appeared to bind at the same site. Cyanide ion produced a much more effective inhibition of renal dipeptidase than the other monoanions, and it was shown that two cyanide ions were bound per enzyme molecule. An investigation of the effect of pH upon monoanion inhibition suggested that the anion inhibitors bind to the group with a pK of approximately 7.8. Complete dissociation of this group (approximately pH 8.4) eliminates the inhibitory effect of anions. The 35Cl line broadening produced by renal dipeptidase in 0.5 M NaCl solutions was 100 times more effective than that produced by equivalent concentrations of aquozinc(II). The line broadening was dependent upon the concentration of the metalloenzyme and independent of the frequency of the exciting radiation. When zinc ion was removed from the metalloenzyme by dialysis or when chloride was titrated from the metalloenzyme by cyanide, line broadening was decreased. Treatment of renal dipeptidase with saturating concentrations of the competitive inhibitor, guanosine triphosphate, in the presence of 0.5 M NaCl also produced a significant decrease in the 35Cl line width. The 35Cl line broadening produced by renal dipeptidase was shown to decrease with increasing pH through the range pH 5.8-10.8. This line-width variation with pH appeared to result from the titration of a site on the metalloprotein with an approximate pK of 7.4. Temperature studies of 35Cl line broadening by the metalloenzyme in the presence of chloride and cyanide inhibitors suggest that the fast exchange process pertains and that the dominant relaxation mechanism is quadrupolar in nature.     

The mitochondrial inner membrane anion channel. Regulation by divalent cations and protons

It is now well established that incubation of mitochondria at pH 8 or higher opens up an electrophoretic anion transport pathway in the inner membrane. It is not known, however, whether this transport process has any physiological relevance. In this communication we demonstrate that anion uniport can take place at physiological pH if the mitochondria are depleted of matrix divalent cations with A23187 and EDTA. Using the light-scattering technique we have quantitated the rates of uniport of a wide variety of anions. Inorganic anions such as Cl-, SO4(2-), and Fe(CN)6(4-) as well as physiologically important anions such as HCO3-, Pi-, citrate, and malate are transported. Some anions, however, such as gluconate and glucuronate do not appear to be transported. On the basis of the finding that the rate of anion uniport assayed in ammonium salts exhibits a dramatic decline associated with loss of matrix K+ via K+/H+ antiport, we suggest that anion uniport is inhibited by matrix protons. Direct inhibition of anion uniport by protons in divalent cation-depleted mitochondria is demonstrated, and the apparent pK of the binding site is shown to be about 7.8. From these properties we tentatively conclude that anion uniport induced by divalent cation depletion and that induced by elevated pH are catalyzed by the same transport pathway, which is regulated by both matrix H+ and Mg2+.     

6-phosphofructokinase (pyrophosphate). Properties of the enzyme from Entamoeba histolytica and its reaction mechanism.

The inorganic pyrophosphate-requiring 6-phosphofructokinase of Entamoeba histolytica has been further investigated. The molecular weight of the enzyme is approximately 83,000 and its isoelectric point occurs at pH 5.8 to 6.0. The divalent cation requirement for reaction was explored. In the direction of fructose 6-phosphate formation half-maximal rate required 500 muM magnesium ion; in the direction of fructose bisphosphate formation 8 muM magnesium ion sufficed. ATP, PPi, polyphosphate, acetyl phosphate, or carbamyl phosphate cannot replace PPi as phosphate donor for the conversion of fructose 6-phosphate to fructose bisphosphate. In the direction of fructose 6-phosphate formation arsenate can replace orthophosphate. Isotope exchange studies indicate that little or no exchange occurs between Pi and PPi or between fructose 6-phosphate and fructose bisphosphate in the absence of a third substrate. These findings appear to rule out phosphoenzyme formation and a ping-pong reaction mechanism. PPi, Pi, and fructose bisphosphate are competitive inhibitors of fructose bisphosphate, PPi, and fructose 6-phosphate, respectively. This argues against an ordered mechanism and suggests a random mechanism. Fructose 6-phosphate and Pi were noncompetitive with respect to each other indicating the formation of a dead end complex. These product inhibition relationships are in accord with a Random Bi Bi mechanism.     

The nature of the link between potassium transport and phosphate transport in Escherichia coli.

A series of mutants of Escherichia coli, combining defects in either of the two phosphate transport systems with defects in one or more of the potassium transport systems, was used to study the nature of the previously observed obligatory requirement for each one of these ions in the transport of the other. The results show that no pair of systems is obligatorily linked, and that either ion can be transported by any one of its systems, provided that a means of entry for the other ion is available. Furthermore, in the total absence of Pi, K+ entry accompanies the transport of other anions, such as aspartate, glutamate, sn-glycero-3-phosphate and glucose 6-phosphate. The results indicate that Pi and the other anions enter by symport with protons, and that a simultaneous K+/H+ exchange, which would serve to maintain the intracellular pH, is responsible for the observed K+ 'symport' with these anions.     

The ATP-Mg/Pi carrier of rat liver mitochondria catalyzes a divalent electroneutral exchange.

Net transport of ATP-Mg or ADP in exchange for phosphate in isolated rat liver mitochondria has been shown to be an electroneutral process mediated by the ATP-Mg/Pi carrier. We compared the steady state distribution ratios of phosphate, ATP-Mg, and ADP at a pH of 7.4 to determine whether the divalent or monovalent form of these anions is the transported substrate. The log of the divalent ATP-Mg distribution ratio (in/out) approached the log of the divalent phosphate distribution ratio (approximately 0.85), which was approximately twice the value of the delta pH (approximately 0.40) across the inner mitochondrial membrane. This steady state relationship held under several different conditions, e.g. when the medium ATP concentration was varied or if the phosphate gradient was modified by partial uncoupling with the proton ionophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Unidirectional ADP efflux in exchange for external ADP or ATP-Mg was stimulated by an increase in matrix H+. The log of the trivalent ADP distribution ratio (approximately 1.20) approached 3 times the value of delta pH. All these data are consistent with the model of an electroneutral exchange of divalent phosphate (HPO2-4) for divalent ATP-Mg (ATP-Mg2-) or for divalent protonated ADP (HADP2-). We conclude that this transport mechanism accounts for the adenine nucleotide concentration gradient that normally exists between the matrix and external medium.     

31P and 35Cl nuclear magnetic resonance measurements of anion transport in human erythrocytes

The exchange of anions across the erythrocyte membrane has been studied using 31P nuclear magnetic resonance (NMR) to monitor inorganic phosphate influx and 35Cl NMR to monitor chloride ion efflux. The 31P NMR resonances for intracellular Pi and extracellular Pi could be observed separately by adjusting the initial extracellular pH to 6.4, while the intracellular pH was 7.3. The 35Cl NMR resonance for intracellular Cl- was so broad as to be virtually undetectable (line width greater than 200 Hz), while that of extracellular Cl-is relatively narrow (line width of about 30 Hz). The transports of Pi and Cl-were both totally inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, a potent inhibitor of the band 3 protein. Since the 31P resonance of Pi varies with pH, intra- and extracellular pH changes could also be determined during anion transport. The extracellular pH rose and intracellular pH fell during anion transport, consistent with the protonated monoanionic H2PO4-form of Pi being transported into the erythrocyte rather than the deprotonated dianionic HPO24-form. The rates of Cl-efflux and Pi influx were determined quantitatively and were found to be in close agreement with values determined by isotope measurements. The Cl-efflux was found to coincide with the influx of the monoanionic H2PO4-form of Pi.     

Sulfate potentiation of the chloride activation of angiotensin converting enzyme

The angiotensin converting enzyme (ACE)-catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions, notably chloride. This activation is enhanced by sulfate; at pH 7.5, the effect is maximal at 0.8 M sulfate and is mediated through a specific interaction of the divalent anion with the enzyme, not through an increase in ionic strength. Sulfate decreases the apparent binding constant for chloride which manifests as a decrease of the apparent KM value, but it does not change kcat. Thus, at pH 7.5, sulfate solely affects substrate binding in accord with the ordered bireactant mechanism of chloride activation that pertains with this substrate [Bünning, P., & Riordan, J.F. (1983) Biochemistry 22, 100-116]. Increasing the pH from 6 to 9 in the absence of sulfate increases the apparent binding constant for chloride almost 60-fold from 3.3 to 190 mM. In the presence of 0.8 M sulfate, however, the change is only about 6-fold, from 0.7 to 4.2 mM. Over the same pH range, the apparent KM for furanacryloyl-Phe-Gly-Gly obtained with saturating chloride concentrations shifts from 0.14 to 0.48 mM, while in the presence of 0.8 M sulfate about 3-fold lower apparent KM values are obtained. Sulfate does not appear to affect the pK of a group on the enzyme that controls the mechanism of chloride activation but rather decreases the apparent KM by reducing the apparent binding constant for chloride.     

The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: II. Kinetic properties of NBD-taurine transfer in asymmetric conditions

The transport of inorganic anions across human red blood cell membranes is accomplished by a carrier-like mechanism which involves an electroneutral and obligatory one-for-one anion exchange. The transport kinetics were described by models that involve alternation of single transport sites between the two membrane surfaces. These models predict that each carrier shows either an inward-facing Ei or an outward-facing Eo, conformation, each capable of binding either a monovalent anion or a divalent anion + a proton, to yield an electroneutral translocating complex. Unidirectional transport rates provide, therefore, a measure for the relative concentration of carriers at a given membrane surface. In the present work we assessed how modulation of the transmembrane distribution of carriers by the anion composition of cells and media, and by pH, affect the anion transport system. We have set the system in asymmetric conditions with respect to anions, so that a fast transportable anion (e.g., chloride) was present in one side of the membrane and slow transportable anions (e.g., sulfate, phosphate, oxalate, isethionate, gluconate, HEPES) were present on the other side of the membrane. The skewed distribution of carriers induced in these conditions were assessed by two methods: 1) NBD-taurine transfer which provided a measure for [Ei], the monovalent inward-facing form of the carrier, and 2) inhibition of NBD-taurine transfer by the specific impermeant and competitive inhibitor 4,4'-dinitro-2,2'-stilbene disulfonic acid (DNDS), which provided a measure for the availability of the carrier at the outer membrane surface. In the various symmetric and asymmetric conditions, we found marked differences in transport rates and transport profiles as well as in the susceptibility of the system to inhibition by DNDS. Direct binding studies of DNDS to cells in the various asymmetric conditions supported the conclusion derived from transport studies that transport sites can be recruited towards the membrane surface facing the slow transportable anions.