A Ligand-dependent Conformational Change of the Na+/Galactose Cotransporter of Vibrio parahaemolyticus,Monitored by Tryptophan Fluorescence

Purification and reconstitution of the active Vibrio parahaemolyticus Na+/galactose transporter (vSGLT) enables us to do protein chemistry studies on a representative member of this class of membrane transporters. By measuring intrinsic tryptophan (Trp) fluorescence, conformational changes on the binding of substrates could be investigated. Trp fluorescence increased by 6% on the addition of saturating levels of both Na+ and D-galactose, with a K0.5 for D-galactose of 0.6 mM. No change was seen on the addition of Na+ alone or by adding D-galactose in the presence of K+. The Trp fluorescence could be quenched by acrylamide, but not by Cs+or I?. In the presence of Na+ or K+ alone, of Na+ or K+ and D-galactose, of Na+ and L-glucose, or in the absence of ligands, the fluorescence quenches by acrylamide were similar. This indicated that the tryptophan exposure to acrylamide was unchanged in the presence or absence of ligands. No shifts in lem maximum were observed. To find the Trp responsible for the change in fluorescence, Trp 448 in transmembrane helix 11 in the putative sugar-binding pocket was mutated. It was found that W448F showed a similar change in Trp fluorescence upon the addition of D-galactose in the presence of Na+. We conclude that the Trp fluorescence properties of the purified and reconstituted Na+/galactose cotransporter are selectively changed by the transported substrates Na+ and D-galactose, but it is not the Trp (W448) in the sugar translocation pathway that is involved.     

High-yield functional expression of human sodium/d-glucose cotransporter1 in Pichia pastoris and characterization of ligand-induced conformational changes as studied by tryptophan fluorescence

Studies on the structure-function relationship of transporters require the availability of sufficient amounts of the protein in a functional state. In this paper, we report the functional expression, purification, and reconstitution of the human sodium/d-glucose cotransporter1 (hSGLT1) in Pichia pastoris and ligand-induced conformational changes of hSGLT1 in solution as studied by intrinsic tryptophan fluorescence. hSGLT1 gene containing FLAG tag at position 574 was cloned into pPICZB plasmid, and the resulting expression vector pPICZB-hSGLT1 was introduced into P. pastoris strain GS115 by electroporation. Purification of recombinant hSGLT1 by nickel-affinity chromatography yields about 3 mg of purified recombinant hSGLT1 per 1-liter of cultured Pichia cells. Purified hSGLT1 migrates on SDS-PAGE with an apparent mass of 55 kDa. Kinetic analysis of hSGLT1 in proteoliposomes revealed sodium-dependent, secondary active, phlorizin-sensitive, and stereospecific alpha-methyl-d-glucopyranoside transport, demonstrating its full catalytic activity. The position of the maximum intrinsic tryptophan fluorescence and titration with hydrophilic collisional quenchers KI, acrylamide, and trichloroethanol suggested that most of Trps in hSGLT1 in solution are in a hydrophobic environment. In the presence of sodium, sugars that have been identified earlier as substrate for the transporter increase intrinsic fluorescence in a saturable manner by a maximum of 15%. alpha-Methyl-d-glucopyranoside had the highest affinity (K(d) = 0.71 mM), followed by d-glucose, d-galactose, d-mannose, and d-allose which showed a much lower affinity. l-Glucose was without effect. d-Glucose also increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary phlorizin, the well-established inhibitor of SGLT1, decreased intrinsic fluorescence by a maximum of 50%, and induced a blue shift of maximum (5 nm). Again, the effects were sodium-dependent and saturable and a high affinity K(d) of 5 muM was observed. In addition the surface of hSGLT1 was labeled with 1-anilinonaphthalene-8-sulfonic acid, a reporter molecule for the surface hydrophobicity. In the presence of sodium, addition of d-glucose decreased ANS fluorescence whereas phlorizin increased ANS fluorescence. Thus three conformational states of SGLT1 could be defined which differ in their packing density and hydrophobicity of their surface. They reflect properties of the empty carrier, the d-glucose loaded carrier facing the outside of membrane and the complex of the outside-orientated carrier with phlorizin.     

Examination of sodium/glucose cotransport by using a visible glucose analogue

The glucose derivative, 2,2,6,6-tetramethylpiperidine-1-oxylglucose (TEMPO-glucose) was synthesized and examined for its ability to substitute for glucose as a substrate for the intestinal brush border membrane Na+/glucose cotransporter. TEMPO-glucose inhibited Na(+)-dependent phlorizin binding with an apparent KI of 18 microM and Na(+)-dependent glucose uptake with an apparent KI of 70 microM. The transport competence of TEMPO-glucose was examined by using two measures of transport. The first involved comparing the reversal of trans Na+ inhibition by D-glucose and TEMPO-glucose. The second directly examined Na(+)-dependent TEMPO-glucose uptake by using TEMPO-glucose quenching of intravesicular fluorescein sulfonate fluorescence. Tryptophan fluorescence was sensitive to TEMPO-glucose in a Na(+)-dependent, glucose-inhibitable manner. The bulk of these tryptophans appeared to be located in hydrophobic environments based on Cs(+)-insensitivity. With the reconstituted cotransporter, TEMPO-glucose, and tryptophan quench reagents, the cotransporter was compared in three transport modes: zero trans uptake, zero trans uptake in the presence of a shunt of membrane potential, and substrate exchange. The results suggest that the cotransporter conformation varies depending on its mode of operation and that TEMPO-glucose may be a useful probe for localizing amino acid residues involved in glucose transport.     

Binding of phlorizin to the isolated C-terminal extramembranous loop of the Na+/glucose cotransporter assessed by intrinsic tryptophan fluorescence

Phlorizin, a phloretin 2'-glucoside, is a potent inhibitor of the Na(+)/glucose cotransporter (SGLT1). On the basis of transport studies in intact cells, a binding site for phlorizin was suggested in the region between amino acids 604-610 of the C-terminal loop 13. To further investigate phlorizin binding titration experiments of the intrinsic Trp fluorescence of isolated wild-type loop 13 and two mutated loops (Y604K and G609K) were carried out. Phlorizin (135 microM) produced approximately 40% quenching of the fluorescence of wild-type loop 13; quenching could also be observed with the two mutated loops. The apparent K(d) was lowest for the wild-type loop 13 (K(d) approximately 23 microM), followed by mutant G609K (57 microM) and mutant Y604K (70 microM). Binding of phlorizin was further confirmed by a decrease of the accessibility of loop 13 to the collisional quencher acrylamide. The interaction involves the aromatic moiety of the aglucone since phloretin (the aglucone of phlorizin) showed almost the same effects as phlorizin, while d-glucose did not. MALDI-TOF experiments revealed that loop 13 contained a disulfide bond between Cys 560 and Cys 608 that is very important for phlorizin-dependent fluorescence quenching. These studies provide direct evidence that loop 13 is a site (important amino acids including 604-609) for the molecular interaction between SGLT1 and phlorizin. They confirm that the aglucone part of the glucoside is responsible for this interaction.     

Sodium-induced conformational changes in the glucose transporter of intestinal brush borders

Using brush-border membranes prepared from rabbit small intestine by Ca2+ precipitation and KSCN treatment, we have studied the kinetics and conformational changes of the glucose carrier. Na+ behaves as a competitive activator of glucose transport under zero-trans conditions. Phenyl isothiocyanate and fluorescein isothiocyanate (FITC) inhibit Na+-dependent transport in an irreversible but substrate-protectable manner. Vesicles pretreated with phenyl isothiocyanate in the presence of substrates were then selectively labeled at the glucose carrier with FITC. Competition experiments with Na+ and phlorizin or glucose indicated that FITC binds to the glucose site on the carrier. Carrier-bound FITC displays a saturable quenching of fluorescence in the presence of Na+. The K0.5 of the Na+-specific quench is 25 mM, which is similar to the apparent Km for Na+ activation of glucose transport. Two tyrosine group-specific reagents, N-acetylimidazole and tetranitromethane, inhibit glucose uptake and fluorescent quenching in a Na+-protectable fashion. FITC labeled a 75-kilodalton peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a substrate-sensitive manner. We conclude that Na+ binds to the glucose symporter of intestinal brush borders, a 75-kilodalton peptide, and this induces a rapid conformation change in the transporter which increases its affinity for D-glucose.     

Fluorescence quenching of human orosomucoid. Accessibility to drugs and small quenching agents.

The fluorescence behaviour of human orosomucoid was investigated. The intrinsic fluorescence was more accessible to acrylamide than to the slightly larger succinimide, indicating limited accessibility to part of the tryptophan population. Although I- showed almost no quenching, that of Cs+ was enhanced, and suggested a region of negative charge proximal to an emitting tryptophan residue. Removal of more than 90% of sialic acid from the glycan chains led to no change in the Cs+, I-, succinimide or acrylamide quenching, indicating that the negatively charged region originates with the protein core. Quenching as a function of pH and temperature supported this view. The binding of chlorpromazine monitored by fluorescence quenching, in the presence and in the absence of the small quenching probes (above), led to a model of its binding domain on orosomucoid that includes two tryptophan residues relatively shielded from the bulk solvent, with the third tryptophan residue being on the periphery of the domain, or affected allotopically and near the negatively charged field.     

Evidence for tyrosyl residues at the Na+ site on the intestinal Na+/glucose cotransporter

A tyrosine group has been identified at, or near, the Na+-binding site of the Na+/glucose and Na+/proline cotransporters of rabbit intestinal brush-borders. Three tyrosine group-specific reagents, n-acetylimidazole, tetranitromethane, and p-nitrobenzene sulfonyl fluoride, were used to evaluate the role of tyrosyl groups in Na+-dependent glucose transport, Na+-dependent phlorizin binding, and the Na+-induced fluorescence quenching of fluorescein isothiocyanate bound to the glucose site of the carrier. All three reagents inhibited glucose transport, phlorizin binding, and fluorescein isothiocyanate quenching by 50-85% with Ki values in the range 7-50 microM. The presence of Na+ during the exposure of membranes to the reagents completely protected against inhibition, the Na+ concentration required to produce 50% protection was 14-36 mM. Fluorescent derivatives of n-acetylimidazole were synthesized to identify the tyrosyl residues on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A total of five polypeptide bands were labeled with eosin or fluorescein n-acetylimidazole in a Na+-sensitive manner. Two of these bands, previously identified as the glucose (75,000-dalton) and proline (100,000-dalton) binding sites of the glucose and proline carriers, account for 50% of the Na+-sensitive tyrosyl residues. On the basis of these studies, we believe that the Na+/glucose cotransporter contains both the Na+ and glucose active sites on the same polypeptide or that the cotransporter consists of two similar polypeptides, each containing one substrate binding site.     

Evidence that inhibitors of anion exchange induce a transmembrane conformational change in band 3

The transport inhibitor, eosin 5-maleimide, reacts specifically at an external site on the membrane-bound domain of the anion exchange protein, Band 3, in the human erythrocyte membrane. The fluorescence of eosin-labeled resealed ghosts or intact cells was found to be resistant to quenching by CsCl, whereas the fluorescence of labeled inside-out vesicles was quenched by about 27% at saturating CsCl concentrations. Since both Cs+ and eosin maleimide were found to be impermeable to the red cell membrane and the vesicles were sealed, these results indicate that after binding of the eosin maleimide at the external transport site of Band 3, the inhibitor becomes exposed to ions on the cytoplasmic surface. The lifetime of the bound eosin maleimide was determined to be 3 ns both in the absence and presence of CsCl, suggesting that quenching is by a static rather than a dynamic (collisional) mechanism. Intrinsic tryptophan fluorescence of erythrocyte membranes was also investigated using anion transport inhibitors which do not appreciably absorb light at 335 nm. Eosin maleimide caused a 25% quenching and 4,4'-dibenzamidodihydrostilbene-2,2'-disulfonate) caused a 7% quenching of tryptophan fluorescence. Covalent labeling of red cells by either eosin maleimide or BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) caused an increase in the susceptibility of membrane tryptophan fluorescence to quenching by CsCl. The quenching constant was similar to that for the quenching of eosin fluorescence and was unperturbed by the presence of 0.5 M KCl. Neither NaCl nor Na citrate produced a large change in the relative magnitude of the tryptophan emission. The tryptophan residues that can be quenched by CsCl appear to be different from those quenched by eosin or BIDS and are possibly located on the cytoplasmic domain of Band 3. The results suggest that a conformational change in the Band 3 protein accompanies the binding of certain anion transport inhibitors to the external transport site of Band 3 and that the inhibitors become exposed on the cytoplasmic side of the red cell membrane.     

Conformational dynamics of hSGLT1 during Na+/glucose cotransport

This study examines the conformations of the Na(+)/glucose cotransporter (SGLT1) during sugar transport using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes. The mutant exhibited similar steady-state and presteady-state kinetics as wild-type SGLT1, and labeling of Cys507 by tetramethylrhodamine-6-maleimide had no effect on kinetics. Our strategy was to record changes in charge and fluorescence in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin. In Na(+) buffer, step jumps in membrane voltage elicited presteady-state currents (charge movements) that decay to the steady state with time constants tau(med) (3-20 ms, medium) and tau(slow) (15-70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (DeltaF). The charge vs. voltage (Q-V) and fluorescence vs. voltage (DeltaF-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: zdelta (apparent valence of voltage sensor) approximately 1; and V(0.5) (midpoint voltage) between -15 and -40 mV. Sugar induced an inward current (Na(+)/glucose cotransport), and reduced maximal charge (Q(max)) and fluorescence (DeltaF(max)) with half-maximal concentrations (K(0.5)) of 1 mM. Increasing [alphaMDG](o) also shifted the V(0.5) for Q and DeltaF to more positive values, with K(0.5)'s approximately 1 mM. The major difference between Q and DeltaF was that at saturating [alphaMDG](o), the presteady-state current (and Q(max)) was totally abolished, whereas DeltaF(max) was only reduced 50%. Phlorizin reduced both Q(max) and DeltaF(max) (K(i) approximately 0.4 microM), with no changes in V(0.5)'s or relaxation time constants. Simulations using an eight-state kinetic model indicate that external sugar increases the occupancy probability of inward-facing conformations at the expense of outward-facing conformations. The simulations predict, and we have observed experimentally, that presteady-state currents are blocked by saturating sugar, but not the changes in fluorescence. Thus we have isolated an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na(+)/sugar cotransport (saturating voltage and external Na(+) and sugar concentrations) is the slow release of Na(+) from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation.     

Conformation of adenosine deaminase in complexes with inhibitors: application of selective quenching of fluorescence emission

The effect of inhibitors, 1-deazaadenosine (1-dAdo) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), on the conformation of adenosine deaminase was studied using the method of selective quenching of fluorescence emission by acrylamide, I- and Cs+. Both in free adenosine deaminase and in its complexes with the inhibitors, the wavelength maxima and half-width of the emission characterize the environment of fluorescing tryptophan residues in adenosine deaminase as weak polar with limited access to solvent. The formation of complexes with the ground state inhibitors used did not quench or change the main emission characteristics of tryptophan fluorescence in adenosine deaminase. Small blue shifts of emission maxima were observed upon quenching in all three samples. The Stern-Volmer parameters of tryptophan fluorescence quenching by acrylamide were not essentially influenced by complex formation of the enzyme with the inhibitors: in general, the folding of the enzyme molecule in the complexes is not perturbed. On the contrary, the emission quenching by charged heavy ions, I- and Cs+, in the complexes was hindered in comparison with free adenosine deaminase. In the complex with 1-deazaadenosine, the parameters for quenching by both ions evidence the essential worsening of their interaction with tryptophans. In the complex with erythro-9-(2-hydroxy-3-nonyl)adenine, along with the worse quenching by I-, complete prohibition of quenching by Cs+ was observed. These data indicate that the local environments of fluorescing tryptophan residues is substantially distorted compared with free adenosine deaminase, which leads to their screening from charged heavy ions.     

Examination of the Na+-induced conformational change of the intestinal brush border sodium/glucose symporter using fluorescent probes

The Na+-induced change in conformation of the intestinal brush border glucose carrier has been examined by three procedures. In the first, we have measured the effect of Na+ on the binding of fluorescein isothiocyanate (FITC) to the glucose site; 100 mM Na increased the specific [blocked by D-glucose, p-(chloromercuri)benzenesulfonic acid, and N-acetylimidazole] FITC binding to a 75-kilodalton polypeptide 3-fold. In the second series, we have examined the effect of Na+ on the susceptibility of the fluorescently labeled glucose site [pyrene isothiocyanate (PYTC) labeled] to a hydrophilic quencher (Tl+); 100 mM NaCl increased the fraction of PYTC sites available to Tl+ from 32% to 92% and decreased the apparent quenching constant from 94 to 44 M-1. Finally, in the third series, we probed the distribution of tryptophan residues 15-30 A from the glucose site using a "distant reporter group method", where tryptophan was used an an energy donor to anthracene isothiocyanate bound to the glucose site. Tryptophan quench reagents (I-, Cs+, and acrylamide) were then employed to probe the accessibility of the glucose site tryptophans in the presence and absence of sodium. In the absence of Na+, there were two major classes of glucose tryptophans--exterior surface residues and residues buried in the hydrophobic protein matrix. Na+ caused a redistribution of the donor tryptophans such that a higher percentage were accessible to I- (51% vs. 25%) and fewer were accessible to Cs+ (13% vs. 25%) and acrylamide (27% vs. 57%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium-independent low-affinity D-glucose transport by human sodium/D-glucose cotransporter 1: critical role of tryptophan 561

Although there is no evidence of significant Na-independent glucose flux in tissues naturally expressing SGLT1, previous kinetic and biophysical studies suggest that sodium/d-glucose cotransporter 1 (hSGLT1) can facilitate sodium-independent d-glucose transport and may contain more than one sugar binding site. In this work, we analyze the kinetic properties and conformational states of isolated hSGLT1 reconstituted in liposomes by transport and fluorescence studies in the absence of sodium. In the transport studies with hSGLT1, significant sodium-independent phlorizin inhibitable alpha-methyl d-glucopyranoside (alpha-MDG) uptake was observed which amounted to approximately 20% of the uptake observed in the presence of a sodium gradient. The apparent affinity constant for alpha-MDG was thereby 3.4 +/- 0.5 mM, a value approximately 10-fold higher than that in the presence of sodium. In the absence of sodium, various sugars significantly decreased the intrinsic Trp fluorescence of hSGLT1 in proteoliposomes exhibiting the following sequence of affinities: alpha-MDG > d-glucose approximately d-galactose > 6-deoxy-d-glucose > 2-deoxy-d-glucose > d-allose. Furthermore, significant protection effects of d-glucose or phlorizin against potassium iodide, acrylamide, or trichloroethanol quenching were observed. To locate the Trps involved in this reaction, we generated mutants in which all Trps were sequentially substituted with Phe. None of the replacements significantly affected sodium-dependent uptake. Uptake in the absence of sodium and typical fluorescence changes depended, however, on the presence of Trp at position 561. This Trp residue is conserved in all known SGLT1 forms (except Vibrio parahaemolyticus SGLT) and all SGLT isoforms in humans (except hSGLT3). If all these data are taken into consideration, it seems that Trp-561 in hSGLT1 forms part of a low-affinity sodium-independent binding and/or translocation site for d-glucose. The rate of sodium-independent translocation via hSGLT1 seems, however, to be tightly regulated in the intact cell by yet unknown factors.     

Functional expression of the Vibrio parahaemolyticus Na+/galactose (vSGLT) cotransporter in Xenopus laevis oocytes

We have successfully expressed a bacterial cotransporter in a functional form in the Xenopus laevis oocyte expression system. The goals were to compare the kinetics and selectivity of the cotransporter expressed in oocytes with those obtained in bacteria and in proteoliposomes, and to determine if it is possible to measure the electrical properties of the bacterial cotransporter expressed in oocytes. The Vibrio parahaemolyticus Na+/galactose cotransporter (vSGLT) expressed in oocytes has functional properties that are similar to those expressed in bacteria and those of the purified cotransporter reconstituted into liposomes. vSGLT is a Na+-dependent transporter that is saturable with Na+ (K(0.5)=17 mM) and D-galactose (K(0.5)=237 microM) and is sensitive to both D-fucose and phlorizin. In addition, vSGLT in oocytes shows a sugar specificity in the order of D-galactose >D-fucose > D-glucose, distinguishing it from the animal members of the Na+/glucose cotransporter family. The level of transport by vSGLT in oocytes is lower overall (V(max) approximately 10 pmol/oocyte/hour) compared to other plant and animal cotransporters (V(max) approximately 1000 pmol/oocyte/hour). The low level of expression does not permit us to carry out electrophysiological studies of the bacterial cotransporter. This study shows the potential and unique advantages of utilizing a eukaryotic oocyte expression system to study bacterial cotransporters.     

Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism.

1. The protein fluorescence intensity of (Na+ + K+)-ATPase is enhanced following binding of K+ at low concentrations. The properties of the response suggest that one or a few tryptophan residues are affected by a conformational transition between the K-bound form E2 . (K) and a Na-bound form E1 . Na. 2. The rate of the conformational transition E2 . (K) leads to E . Na has been measured with a stopped-flow fluorimeter by exploiting the difference in fluorescence of the two states. In the absence of ATP the rate is very slow, but it is greatly accelerated by binding of ATP to a low affinity site. 3. Transient changes in tryptophan fluorescence accompany hydrolysis of ATP at low concentrations, in media containing Mg2+, Na+ and K+. The fluorescence response reflects interconversion between the initial enzyme conformation, E1 . Na and the steady-state turnover intermediate E2 . (K). 4. The phosphorylated intermediate, E2P can be detected by a fluorescence increase accompanying hydrolysis of ATP in media containing Mg2+ and Na+ but no K+. 5. The conformational states and reaction mechanism of the (Na+ + K+)-ATPase are discussed in the light of this work. The results permit a comparison of the behaviour of the enzyme at both low and high nucleotide concentrations.     

Fluorescence characterization of Trp 21 in rat glutathione S-transferase 1–1: Microconformational changes induced by S-hexyl glutathione

The glutathione S-transferase (GST) isoenzyme A1–1 from rat contains a single tryptophan, Trp 21, which is expected to lie within α-helix 1 based on comparison with the X-ray crystal structures of the pi- and mu-class enzymes. Steady-state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand-induced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme A1–1 causes an increase in the steady-state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequency-domain excited-state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substrate-free GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate-free protein, the short lifetime component contributes 9–16% of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-hexyl glutathione. Steady-state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide-quenching experiments at several emission wavelengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the “H-site” is occupied.     

Cation specificity and modes of the Na+:CO3(2-):HCO3- cotransporter in renal basolateral membrane vesicles

The cation specificity and possible exchange modes of the Na+:CO3(2-):HCO3- cotransporter were evaluated by use of basolateral membrane vesicles isolated from rabbit renal cortex. External Li+ inhibited HCO3- gradient-stimulated 22Na uptake, indicating that Li+ interacts with the Na+:CO3(2-):HCO3- cotransporter. No interaction with K+, choline, Rb+, Cs+, or NH4+ could be similarly detected. Imposing an outward Li+ gradient caused quenching of acridine orange fluorescence in the presence but not in the absence of HCO3-, suggesting that Li+:base cotransport takes place via the Na+:CO3(2-):HCO3- cotransporter. Imposing an outward gradient of unlabeled Na+ stimulated the initial rate of 22Na uptake and induced its transient uphill accumulation, indicating Na(+)-Na+ exchange. Na(+)-Na+ exchange was observed in the presence but not in the absence of HCO3- and was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), suggesting that it occurs via the Na+:CO3(2-):HCO3- cotransporter. Similarly, an outward Li+ gradient stimulated uphill 22Na accumulation, indicating Na(+)-Li+ exchange. Na(+)-Li+ exchange was observed in the presence but not in the absence of HCO3-, and was inhibited by DIDS, suggesting that it also occurs via the Na+:CO3(2-):HCO3- cotransporter. Both Na(+)-Na+ and Li(+)-Na+ exchange modes were sensitive to inhibition by harmaline but not by amiloride. We conclude that Li+ is an alternative substrate for the renal Na+:CO3(2-):HCO3- cotransporter. Transport modes of the system include cation:base cotransport and HCO3-dependent cation-cation exchange.     

Luminescent properties of NADPH: adrenodoxin reductase

The fluorescent and phosphorescent properties of NADPH-adrenodoxin reductase were investigated. It was shown that the fluorescence of protein tryptophanyls was quenched completely by acrylamide and partially by ionic quenchers (I- and Cs+). A removal of the prosthetic group from the protein causes insignificant changes in fluorescent properties of the enzyme. The denaturation of the enzyme by urea was accompanied by growth of quenching parameters. Indeed, some differences were observed in the quenching of flavin fluorescence by ionic quenchers (I- and Cs+). NADPH appeared to be an efficient quencher of NADPH-adrenodoxin reductase tryptophan fluorescence. Using F?rster's equations for non-radiative energy transfer, the distance between NADPH-binding site and tryptophanyls was evaluated to 35-40 A.     

Cyclosporin binding to a protein component of the renal Na(+)-D-glucose cotransporter

The immunosuppressive and nephrotoxic agent cyclosporin binds to a renal polypeptide with an apparent molecular weight of 75,000 which has been identified as a component of the renal Na(+)-D-glucose cotransporter (Neeb, M., Kunz, U., and Koepsell, H. (1987) J. Biol. Chem. 262, 10718-10729). The same Mr 75,000 polypeptide was covalently labeled with the D-glucose analog 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside and with the cyclosporin analog N epsilon-(diazotrifluoroethyl)benzyl-D-Lys8- cyclosporin (CSDZ). CSDZ labeling was decreased when the brush-border membrane proteins were incubated with monoclonal antibodies against the Na(+)-D-glucose cotransporter. In the presence of 145 mM Na+, CSDZ labeling was decreased by D-glucose (1 microM, 1 mM, or 100 mM) and by phlorizin (100 or 500 microM). In the absence of Na+, CSDZ labeling was distinctly increased by 50 microM phlorizin and was slightly increased by 1 mM D-glucose, whereas CSDZ labeling was decreased by 50 microM phloretin and by 500 microM phlorizin. Furthermore, Na(+)-dependent high affinity phlorizin binding to the Na(+)-D-glucose cotransporter was competitively inhibited by cyclosporin A (Ki = 0.04 microM) while Na(+)-D-glucose cotransport was not influenced. The data suggest that a part of the cyclosporin binding domain on the Na(+)-D-glucose cotransporter is identical to the phloretin binding domain of the high affinity phlorizin binding site. While phloretin or the phloretin moiety of phlorizin may directly displace cyclosporin, interaction of D-glucose or of the D-glucose moiety of phlorizin with the transporter may alter the conformation of the cyclosporin binding site and this conformational change may be modulated by Na+.