N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) as a photoaffinity probe for identifying membrane components containing the modifier site of the human red blood cell anion exchange system

Exposure of cells to intense light with the photoactivatable reagent, N- (4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), present in the external medium results in irreversible inhibition of chloride or sulfate exchange. This irreversible inhibition seems to result from covalent reaction with the same sites to which NAP-taurine binds reversibly in the dark. As shown in the preceding paper, high chloride concentrations decrease the reversible inhibition by NAP-taurine in the dark, in a manner suggesting that NAP-taurine and chloride compete for the modifier site of the anion transport system. In a similar fashion, high chloride concentrations in the medium during exposure to light cause a decrease in both the irreversible binding of NAP-taurine to the membrane and the inhibition of chloride exchange. Most of the chloride- sensitive irreversibly bound NAP-taurine is found in the 95,000 dalton polypeptide known as band 3 and, after pronase treatment of intact cells, in the 65,000 dalton fragment of this protein produced by proteolytic cleavage. After chymotrypsin treatment of ghosts, the NAP- taurine is localized in the 17,000 dalton transmembrane portion of this fragment. Although the possible involvement of minor labeled proteins cannot be rigorously excluded, the modifier site labeled by external NAP-taurine appears, therefore, to be located in the same portion of the 95,000 dalton polypeptide as is the transport site.     

Effects of the transport site conformation on the binding of external NAP-taurine to the human erythrocyte anion exchange system. Evidence for intrinsic asymmetry

External N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) inhibits human red cell chloride exchange by binding to a site that is distinct from the chloride transport site. Increases in the intracellular chloride concentration (at constant external chloride) cause an increase in the inhibitory potency of external NAP-taurine. This effect is not due to the changes in pH or membrane potential that usually accompany a chloride gradient, since even when these changes are reversed or eliminated the inhibitory potency remains high. According to the ping-pong model for anion exchange, such transmembrane effects of intracellular chloride on external NAP-taurine can be explained if NAP-taurine only binds to its site when the transport site is in the outward-facing (Eo or EClo ) form. Since NAP-taurine prevents the conformational change from EClo to ECli , it must lock the system in the outward-facing form. NAP-taurine can therefore be used just like the competitive inhibitor H2DIDS (4,4'-diisothiocyano-1,2- diphenylethane -2,2'-disulfonic acid) to monitor the fraction of transport sites that face outward. A quantitative analysis of the effects of chloride gradients on the inhibitory potency of NAP-taurine and H2DIDS reveals that the transport system is intrinsically asymmetric, such that when Cli = Clo, most of the unloaded transport sites face the cytoplasmic side of the membrane.     

Relationship of net chloride flow across the human erythrocyte membrane to the anion exchange mechanism

The parallel effects of the anion transport inhibitor DIDS (4,4'- diisothiocyanostilbene-2,2'-disulfonate) on net chloride flow and on chloride exchange suggest that a major portion of net chloride flow takes place through the anion exchange system. The "slippage" model postulates that the rate of net anion flow is determined by the movement of the unloaded anion transport site across the membrane. Both the halide selectivity of net anion flow and the dependence of net chloride flux on chloride concentration over the range of 75 to 300 mM are inconsistent with the slippage model. Models in which the divalent form of the anion exchange carrier or water pores mediate net anion flow are also inconsistent with the data. The observations that net chloride flux increases with chloride concentration and that the DIDS- sensitive component tends to saturate suggest a model in which net anion flow involves "transit" of anions through the diffusion barriers in series with the transport site, without any change in transport site conformation such as normally occurs during the anion exchange process. This model is successful in predicting that the anion exchange inhibitor NAP-taurine, which binds to the modifier site and inhibits the conformational change, has less effect on net chloride flow than on chloride exchange.     

Titration of transport and modifier sites in the red cell anion transport system

This work demonstrates the existence of titratable transport and modifier sites in the anion transport system of human red cells. Effects of alkaline extracellular pH on chloride exchange were studied up to pH 13 at 0 degrees C. The studies revealed two sets of reversible titratable groups. One set, having a pK of or approximately 11, appeared to be identical with the inhibitory halide-binding modifier site. Deprotonation of this site stimulated anion transport. The apparent dissociation constants of chloride and iodide at this modifier site were 0.3 and 0.06 M, respectively, and it was confirmed that the organic sulfonate NAP-taurine inhibits anion transport reversibly by a high-affinity interaction with halide-binding modifier sites at the extracellular side of the membrane. Other groups, with apparent pK of or approximately 12 at chloride concentrations above 0.1 M, were named as "transport sites" because transport function depended totally on their protonation. The apparent pK decreased when extracellular halide concentrations was lowered below 0.1 M. It was dependent of the intracellular chloride concentration, and was equally sensitive to extracellular pH of 13, was fully reversible. Hydroxyl ions were not transported to an appreciable extent by the anion exchange system. The pK values of both sets of groups make it likely that they are both arginyl residues, functioning as anion recognition sites similar to the role of functionally essential arginyl residues observed with numerous enzymes.     

Anion transport in red blood cells. II. Kinetics of reversible inhibition by nitroaromatic sulfonic acids.

The anion exchange system of human red blood cells is highly inhibited and specifically labeled by isothiocyano derivatives of benzene sulfonate (BS) or stilbene disulfonate (DS). To learn about the site of action of these irreversibly binding probes we studied the mechanism of inhibition of anion exchange by the reversibly binding analogs p-nitrobenzene sulfonic acid (pNBS) and 4,4'-dinitrostilbene-disulfonic acid (DNDS). In the absence of inhibitor, the self-exchange flux of sulfate (pH 7.4, 25 degrees C) at high substrate concentration displayed self-inhibitory properties, indicating the existence of two anion binding sites: one a high-affinity transport site and the other a low-affinity modifier site whose occupancy by anions results in a noncompetitive inhibition of transport. The maximal sulfate exchange flux per unit area was JA = (0.69 +/- 0.11) X 10(-10) moles . min-1 . cm-2 and the Michaelis-Menten constants were for the transport site KS = 41 +/- 14 mM and for the modifier site Ks' = 653 +/- 242 mM. The addition to cells of either pNBS at millimolar concentrations or DNDS at micromolar concentrations led to reversible inhibition of sulfate exchange (pH 7.4, 25 degrees C). The relationship between inhibitor concentration and fractional inhibition was linear over the full range of pNBS or DNDS concentrations (Hill coefficient n approximately equal to 1), indicating a single site of inhibition for the two probes. The kinetics of sulfate exchange in the presence of either inhibitor was compatible with that of competitive inhibition. Using various analytical techniques it was possible to determine that the sulfate transport site was the target for the action of the inhibitors. The inhibitory constants (Ki) for the transport sites were 0.45 +/- 0.10 microM for DNDS and 0.21 +/- 0.07 mM for pNBS. From the similarities between reversibly and irreversibly binding BS and DS inhibitors in structures, chemical properties, modus operandi, stoichiometry of interaction with inhibitory sites, and relative inhibitory potencies, we concluded that the anion transport sites are also the sites of inhibition and of labeling of covalent binding analogs of BS and DS.     

Transmembrane effects of irreversible inhibitors of anion transport in red blood cells. Evidence for mobile transport sites

Experiments were designed to determine whether band 3, the anion transport protein of the red cell membrane, contains a mobile element that acts as a carrier to move the anions across a permeability barrier. The transport site-specific, nonpenetrating irreversible inhibitor 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS) was found to be effective only when applied extracellularly. It was used to sequester transport sites on the extracellular side of the membrane in intact cells. The membranes were then coverted into inside-out vesicles. The number of anion transport sites available on the cytoplasmic side of the vesicle membranes was then estimated by measuring the binding of N-(-4-azido-2-nitrophenyl)-2-aminoethyl-sulfonate (NAP-taurine), a photoreactive probe. Pretreatment with DIDS from the extracullular side substantially reduced the binding of NAP-taurine at the cytoplasmic side. Since NAP-taurine does not appear to penetrate into the intravesicular (normally extracellular) space, a transmembrane effect is apparently involved. About 70% of the DIDS-sensitive NAP-taurine binding sites are located in band 3, with the remainder largely in a lower molecular weight (band 4) region. A similar pattern of reduction in NAP-taurine binding is produced by high concentrations of Cl-, but this anion has little or no effect in vesicles from cells pretreated with DIDS. Thus the DIDS-modulated sites seem to be capable of binding either NAP-taurine or Cl. It is suggested that band 3 contains a mobile transport element that can be recruited to the extracellular surface by DIDS, thus becoming unavailable to NAP-taurine at the cytoplasmic face of the membrane. The results are consistent with a model of carrier-mediated transport in which the movement of the transport site is associated with a local conformational change in band 3 protein.     

Use of niflumic acid to determine the nature of the asymmetry of the human erythrocyte anion exchange system

Niflumic acid is a noncompetitive inhibitor of chloride exchange, which binds to a site different from the transport or modifier sites. When the internal Cl- concentration is raised, at constant extracellular Cl- , the inhibitory potency of niflumic acid increases. This effect cannot be attributed to changes in membrane potential, but rather it suggests that niflumic acid binds to the anion exchange protein band 3 only when the transport site faces outward. When the chloride gradient is reversed, with Clo greater than Cli , the inhibitory potency of niflumic acid decreases greatly, which indicates that the affinity of niflumic acid for band 3 with the transport site facing inward is almost 50 times less than when the transport site faces outward. Experiments in which Cli = Clo show no significant change in the inhibition by niflumic acid when Cl- is lowered from 150 to 10 mM. These data suggest that the intrinsic dissociation constants for Cl- at the two sides of the membrane are nearly equal. Thus, the chloride- loaded transport sites have an asymmetric orientation like that of the unloaded transport sites, with approximately 15 times more sites facing the inside than the outside. The asymmetry reflects an approximately 1.5 kcal/mol free energy difference between the inward-facing and outward-facing chloride-loaded forms of band 3. High concentrations of chloride (with Cli = Clo), which partially saturate the modifier site, have no effect on niflumic acid inhibition, which indicates that chloride binds equally well to the modifier site regardless of the orientation of the transport site.     

Inhibition of terminal deoxynucleotidyltransferase by (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate

(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate (BVdUTP), known as a specific inhibitor of herpes simplex virus (type 1)-DNA polymerase, was found to be a potent inhibitor of the activity of terminal deoxynucleotidyltransferase (TdT) from calf thymus. BVdUTP was not an efficient substrate of TdT, but it inhibited the incorporation of normal deoxynucleotide substrates in competitive fashion at the nucleotide binding site of TdT molecule. The Ki value for BVdUTP (5 microM) was much less than the Km value for dGTP (83 microM), indicating stronger affinity of the inhibitor to TdT than that of the substrate. These results indicate the usefulness of BVdUTP as a potent inhibitor of TdT for elucidation of the reaction mechanism of this enzyme.     

The anion-transfer system of erythrocyte membranes. N-(7-Nitrobenzofurazan-4-yl)taurine, a fluorescent substrate-analogue of the system.

The fluorescent probe Nbd-Tau [N-(7-nitrobenzofurazan-4-yl)taurine] was synthesized and evaluated as a potential substrate of the anion-transport system of human erythrocyte membrane. The probe inhibited Cl- exchange in a competitive manner from either surface of the membrane, displaying Ki values in the mM range at the inner surface and in the microM range at the outer surface. Inhibition from within cells was via interaction with Cl--transport sites, whereas from it was via interaction with sites of unidentified nature. Nbd-Tau efflux from cells was monitored fluorimetrically in a continuous mode by a novel method that circumvents separation of the cells from the medium. Using this method, it is shown that Nbd-Tau efflux fulfils the following criteria of a substrate of the anion transport system: (a) susceptibility to classical and specific inhibitors of the system; (b) competitive inhibition with Cl- for anion-transport sites; and (c) temperature coefficient comparable with that of Cl- exchange. The fluorometric method is highly sensitive, versatile, and kinetically informative. With minor modifications it can be used for measuring anion transport across "ghost" and isolated membrane vesicles.     

Anion Transport in Red Blood Cells II. Kinetics of Reversible Inhibition by Nitroaromatic Sulfonic Acids

The anion exchange system of human red blood cells is highly inhibited and specifically labeled by isothiocyano derivatives of benzene sulfonate (BS) or stilbene disulfonate (DS). To learn about the site of action of these irreversibly binding probes we studied the mechanism of inhibition of anion exchange by the reversibly binding analogs p-nitrobenzene sulfonic acid (pNBS) and 4,4′-dinitrostilbene-disulfonic acid (DNDS). In the absence of inhibitor, the self-exchange flux of sulfate (pH 7.4, 25°C) at high substrate concentration displayed self-inhibitory properties, indicating the existence of two anion binding sites: one a high-affinity transport site and the other a low-affinity modifier site whose occupancy by anions results in a noncompetitive inhibition of transport. The maximal sulfate exchange flux per unit area was J_A = (0.69 ± 0.11) × 10^-10 moles · min^-1 · cm^-2 and the Michaelis-Menten constants were for the transport site K_S = 41 ± 14 mM and for the modifier site K_S' = 653 ± 242 mM. The addition to cells of either pNBS at millimolar concentrations or DNDS at micromolar concentrations led to reversible inhibition of sulfate exchange (pH 7.4, 25°C). The relationship between inhibitor concentration and fractional inhibition was linear over the full range of pNBS or DNDS concentrations (Hill coefficient n ? 1), indicating a single site of inhibition for the two probes. The kinetics of sul- fate exchange in the presence of either inhibitor was compatible with that of competitive inhibition. Using various analytical techniques it was possible to determine that the sulfate trans- port site was the target for the action of the inhibitors. The in- hibitory constants (Ki j for the transport sites were 0.45 ± 0.10 PM for DNDS and 0.21 ± 0.07 mM for pNBS. From the similarities between reversibly and irreversibly binding BS and DS inhibitors in structures, chemical properties, modus oper- andi, stoichiometry of interaction with inhibitory sites, and relative inhibitory potencies, we concluded that the anion trans- port sites are also the sites of inhibition and of labeling of co- valent binding analogs of BS and DS.     

Chinese hamster ovary cell mutants deficient in an anion exchanger functionally similar to the erythroid band 3

Studies in Chinese hamster ovary cells demonstrate the presence of an anion exchanger, which has some of the properties of the band 3 transporter in erythrocytes. 1) Extracellular chloride is a competitive inhibitor of sulfate influx and stimulates sulfate efflux, suggesting that the mechanism of uptake is SO2-(4)/Cl- exchange. 2) The anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits sulfate uptake in a dose-dependent manner. Half-maximal inhibition is achieved at 0.06 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. 3) Low extracellular pH markedly stimulates sulfate uptake. A 6-fold decrease in the apparent Km is observed at pHout 5.5 as compared to pHout 7.5. However, studies carried out over a broad range of extracellular SO2-(4) concentrations indicate the presence of three components of this transport activity in Chinese hamster ovary cells: two high affinity low capacity systems, one in the range 0.5 microM less than [SO2-(4)]out less than 50 microM and one in the range 50 microM less than [SO2-(4)]out less than 150 microM, and a low affinity, high capacity system (at [SO2-(4)]out greater than 150 microM). These properties have not been previously reported for the erythroid band 3 transporter. The availability of mutants deficient in these activities has enabled us to carry out studies which suggest that the high affinity systems are functionally independent of the low affinity system, but that all systems are dependent on the same anion exchange protein. Studies in a mutant which lacks all components of the transport activity indicates that the anion exchanger may be instrumental in the regulation of the intracellular pH in Chinese hamster ovary cells.     

Effects of 10-(2-propynyl)-estr-4-ene-3,17-dione (MDL 18,962)--a mechanism-based irreversible inhibitor of aromatase--in cultured human foreskin fibroblasts

In male subjects, peripheral aromatization of androgens accounts for most of the estrogen production, and skin is an important site of such enzymatic activity. We have studied the effects of a mechanism-based, irreversible aromatase inhibitor, 10-(2-propynyl)-estr-4-ene-3,17-dione (MDL 18,962) on androgen action and metabolism in cultured human foreskin fibroblasts. Cells were incubated simultaneously in the presence of substrate, androstenedione, and inhibitor, MDL 18,962. Aromatase activity was linear with time up to 3 h of incubation at 37 degrees C in the absence and presence of 1.0-10 nM inhibitor. The IC50 for four different cell strains ranged from 4.0 to 8.6 nM MDL 18,962. Kinetic analysis of competitive inhibition by the Eadie-Hofstee method yielded an apparent Ki of 2.75 nM for the inhibitor. Preincubation of cells with MDL 18,962 resulted in irreversible inhibition of aromatase activity which was time- and concentration-dependent. We calculated a Ki of 7.6 nM for MDL 18,962. Preincubation of cells with 25 nM MDL 18,962 suppressed enzyme activity for up to 6 h following removal of the inhibitor, before a return of enzyme activity due to synthesis of new enzyme. MDL 18,962 (0.2-20 microM) did not influence the 5 alpha-reduction of testosterone (200 nM). In addition, binding of dihydrotestosterone (2 nM) to androgen receptors was not affected by MDL 18,962 (25-1000 nM). In summary, MDL 18,962 is a specific, high potency inhibitor of aromatase. By virtue of its high binding affinity to the enzyme active site, it competes very effectively with substrate, resulting in irreversible inactivation of aromatase.     

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.     

S-(N-dansylaminoethyl)-6-mercaptoguanosine as a fluorescent probe for the uridine transport system in human erythrocytes.

A fluorescent derivative of 6-mercaptoguanosine, S-(N-dansylaminoethyl)-6-mercaptoguanosine, was synthesized, and found to be a strong inhibitor of the uridine transport system of erythrocyte (Ki approximately 0.3 microM). The emission spectrum of this compound has peaks at 400 and 550 nm. The emission at 550, but not that a 400 nm, in environment-sensitive. A method was devised for preparing a suspension of erythrocyte-membrane fragments with sufficiently low light scattering so that a detailed study could be made of the fluorescence of the probe when bound to membranes. Direct binding measurements showed the existence of a tight binding site, with a dissociation constant of the same order of magnitude as the inhibition constant. Binding of probe and substrate are not mutually exclusive, but the fluorescence and affinity of the bound probe are sensitive to the presence of uridine. The emission spectrum suggests that the bound probe penetrates into the bilayer region of the membrane.     

Reaction of the glucose carrier of erythrocytes with sodium tetrathionate: Evidence for inward-facing and outward-facing carrier conformations

Summary Sodium tetrathionate reacts with the glucose carrier of human erythrocytes at a rate which is greatly altered in the presence of competitive inhibitors of glucose transport. Inhibitors bound to the carrier on the outer surface of the membrane, either at the substrate site (maltose) or at the external inhibition site (phloretin and phlorizin), more than double the reaction rate. Inhibitors bound at the internal inhibition site (cytochalasin B and androstenedione), protect the system against tetrathionate. After treatment with tetrathionate, the maximum transport rate falls to less than one-third, and the properties of the binding sites are modified in unexpected ways. The affinity of externally bound inhibitors rises: phloretin is bound up to seven times more strongly and phlorizin and maltose twice as strongly. The affinity of cytochalasin B, bound at the internal inhibition site, falls to half while that of androstenedione is little changed. The affinity of external glucose falls slightly. Androstenedione prevents both the fall in transport activity and the increase in phloretin affinity produced by tetrathionate. An inhibitor of anion transport has no effect on the reaction. The observations support the following conclusions: (1) Tetrathionate produces its effects on the glucose transport system by reacting with the carrier on the outer surface of the membrane. (2) The carrier assumes distinct inward-facing and outward-facing conformations, and tetrathionate reacts with only the outward-facing form. (3) The thiol group with which tetrathionate is presumed to react is not present in either the substrate site or the internal or external inhibitor site. (4) In binding asymmetrically to the carrier, a reversible inhibitor shifts the carrier partition between inner and outer forms and thereby raises or lowers the rate of tetrathionate reaction with the system. (5) Reaction with tetrathionate converts the carrier to an altered state in which the conformation at all three binding sites is changed and the rate of carrier reorientation is reduced.     

Mixed type inhibition of the renal Na+/H+ antiporter by Li+ and amiloride. Evidence for a modifier site

We studied the interactions of Na+, Li+, and amiloride on the Na+/H+ antiporter in brush-border membrane vesicles from rabbit renal cortex. Cation-mediated collapse of an outwardly directed proton gradient (pHin = 6.0; pHout = 7.5) was monitored with the fluorescent amine, acridine orange. Proton efflux resulting from external addition of Na+ or Li+ exhibited simple saturation kinetics with Hill coefficients of 1.0. However, kinetic parameters for Na+ and Li+ differed (Km for Li+ = 1.2 +/- 0.1 mM; Km for Na+ = 14.3 +/- 0.8 mM; Vmax for Li+ = 2.40 +/- 0.07 fluorescence units/s/mg of protein; Vmax for Na+ = 7.10 +/- 0.24 fluorescence units/s/mg of protein). Inhibition of Na+/H+ exchange by Li+ and amiloride was also studied. Li+ inhibited the Na+/H+ antiporter by two mechanisms. Na+ and Li+ competed with each other at the cation transport site. However, when [Na+] was markedly higher than [Li+], [( Na+] = 90 mM; [Li+] less than 1 mM), we observed noncompetitive inhibition (Vmax for Na+/H+ exchange reduced by 25%). The apparent Ki for this noncompetitive inhibition was congruent to 50 microM. In addition, 2-30 mM intravesicular Li+, but not Na+, resulted in trans inhibition of Na+/H+ exchange. Amiloride was a mixed inhibitor of Na+/H+ exchange (Ki = 30 microM, Ki' = 90 microM) but was only a simple competitive inhibitor of Li+/H+ exchange (Ki = 10 microM). At [Li] = 1 mM and [amiloride] less than 100 microM, inhibition of Na+/H+ exchange by a combination of the two inhibitors was always less than additive. These results suggest the presence of a cation-binding site (separate from the cation-transport site) which could be a modifier site of the Na+/H+ antiporter.     

Feedback inhibition of ammonium (methylammonium) ion transport in Escherichia coli by glutamine and glutamine analogs.

When cultured with glutamate or glutamine as the nitrogen source, Escherichia coli expresses a specific ammonium (methylammonium) transport system. Over 95% of the methylammonium transport activity in washed cells was blocked by incubation with 100 microM L-glutamine in the presence of chloramphenicol (100 micrograms/ml). The time course for the onset of this glutamine inhibition followed a first-order rate expression with a t1/2 of 2.8 min. The inhibition of transport by L-glutamine was noncompetitive (Ki = 18 microM) with respect to the [14C]methylammonium substrate. D-Glutamine had no significant effect. The glutamine analogs gamma-L-glutamyl hydroxamate (Ki = 360 microM) and gamma-L-glutamyl hydrazide (Ki = 800 microM) were also noncompetitive inhibitors of methylammonium transport, suggesting that glutamine metabolism is not required. The role of the intracellular glutamine pool in the regulation of ammonium transport was investigated by using mutants carrying defects in the operon of glnP, the gene for the glutamine transporter. The glnP mutants had normal rates of methylammonium transport but were refractory to glutamine inhibition. Glycylglycine, a noncompetitive inhibitor of methylammonium uptake in wild-type cells (Ki = 43 microM), was equipotent in blocking transport in glnP mutants. Although ammonium transport is also subject to repression by growth of E. coli in the presence of ammonia, this phenomenon is unrelated to glutamine inhibition. A GlnL RegC mutant which constitutively expressed ammonium transport activity exhibited a sensitivity to glutamine inhibition similar to that of wild-type cells. These findings indicate that ammonium transport in E. coli is regulated by the internal glutamine pool via feedback inhibition.     

Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX

Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.     

Selective inhibition of thrombin by (2R,4R)-4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl++ +) sulfonyl]-l-arginyl)]-2-piperidinecarboxylic acid

The potency of thrombin inhibition by 4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-sulfony l]- L-arginyl]-2-piperidinecarboxylic acid (MQPA) depended on the stereoconformation of the 2-piperidinecarboxylic acid moiety. Ki values for bovine alpha-thrombin were 0.019 microM with (2R,4R)-MQPA, 0.24 microM with (2R,4S)-MQPA, 1.9 microM with (2S,4R)-MQPA, and 280 microM with (2S,4S)-MQPA. (2R,4R)-MQPA of the four stereoisomers of MQPA was also the most potent inhibitor for other trypsin-like serine proteases with Ki values of 5.0 microM for trypsin, 210 microM for factor Xa, 800 microM for plasmin, and 1500 microM for plasma kallikrein. Examination of the potency of thrombin inhibition by arginine derivatives related to MQPA in structure suggested the presence of a specific binding site for the carboxamide portion (C-terminal side). The relative inhibitory potency of the four stereoisomers of MQPA for trypsin was nearly identical with that for thrombin, suggesting that the specific binding site for the carboxamide portion is present in both enzymes. Modification of thrombin by phosphopyridoxylation or the presence of heparin did not significantly alter the binding of MQPA.     

Anion transport in red blood cells. III. Sites and sidedness of inhibition by high-affinity reversible binding probes

Studies of binding of the reversible inhibitor DNDS (for abbreviations, see Nomenclature) and red blood cell membranes revealed 8.6 +/- 0.7 x 10(5) high-affinity binding sites per cell (KD = 0.8 +/- 0.4 muM). Under conditions of "mutual depletion," inhibition studies of anion exchange revealed 8.0 +/- 0.7 x 10(5) DNDS inhibitory sites per cell (KD = 0.87 +/- 0.04 muM). Binding and kinetics studies with DNDS indicate that there are 0.8 -- 0.9 x 10(6) functional anion transport sites per blood cell. The transport of DNDS displayed high temperature and concentration dependencies, chemical specificity, susceptibility to inhibition by DIDS, and differences between egress and ingress properties. Under conditions of no DNDS penetration (e.g., 0 degrees C), inhibition of anion exchange by DNDS showed marked sidedness from the outside inhibitions and were demonstrable at micromolar concentrations, whereas from the inside no inhibition occurred even at millimolar concentrations. The asymmetry of DNDS transport properties and the sidedness of binding and inhibition suggest that anion transport sites have a very low affinity for or are inaccessible to DNDS at the inner membrane face. The site of DNDS permeation, although susceptible to DIDS, is apparently not the site of anion exchange.