Oxalate Exchange Research Articles

Projection structure of the bacterial oxalate transporter OxlT at 3.4 Å resolution

OxlT is a bacterial transporter protein with 12 transmembrane segments that belongs to the Major Facilitator Superfamily of transporters. It facilitates the exchange of oxalate and formate across the membrane of the Gram-negative bacterium Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional, tube-like crystals of OxlT, we have previously determined the three-dimensional structure of this transporter at 6.5 Å resolution. Here, we report conditions to obtain crystalline, two-dimensional sheets of OxlT with diameters exceeding 2 μm. Images of the crystalline sheets were recorded at liquid nitrogen temperatures on a transmission electron microscope equipped with a field-emission gun, operated at 300 kV. Computed optical diffraction patterns from the best images display measurable reflections to about 3.4 Å, and electron diffraction patterns show spots to about 3.2 Å resolution in the best cases. As in the case of the tube-like crystals, the new crystalline sheets also belong to the p22 12 1 symmetry group. However, the unit cell dimensions of 102.7 Å × 67.3 Å are significantly smaller in one direction than those previously observed with the tube-like crystals that display unit cell dimensions of 100.3 Å × 79.0 Å. Different regions of OxlT are involved in intermolecular contacts in the two types of crystals, and the improved resolution of the sheet crystals appears to be mainly attributable to this tighter packing of the monomers within the unit cell.

Specificity of Anion Exchange Mediated by Mouse Slc26a6

Recently, CFEX, the mouse orthologue of human SLC26A6, was localized to the brush border membrane of proximal tubule cells and was demonstrated to mediate Cl(-)-formate exchange when expressed in Xenopus oocytes. The purpose of the present study was to examine whether mouse Slc26a6 can mediate one or more of the additional anion exchange processes observed to take place across the apical membrane of proximal tubule cells. Influx of [(14)C]formate into Slc26a6-expressing oocytes was inhibited by sulfate, oxalate, and p-aminohippurate (PAH), indicating affinity for these anions. Measurements of uptake of [(14)C]oxalate, [(14)C]PAH, and [(35)S]sulfate indicated that Slc26a6 can mediate transport of oxalate and sulfate but not PAH. Studies of the effect of external anions on [(14)C]oxalate efflux demonstrated Slc26a6-mediated Cl(-)-oxalate, oxalate-formate, oxalate-oxalate, and oxalate-sulfate exchange. Two-electrode voltage clamp measurements indicated that Slc26a6-mediated Cl(-)-oxalate exchange is electrogenic. Intracellular pH recordings demonstrated that Slc26a6 can mediate Cl(-)-HCO(3)(-) exchange, but Cl(-)-OH(-) exchange was not detected. The presence of 100 microm oxalate inhibited the rate of Cl(-)-HCO(3)(-) exchange by 60%. We conclude that mouse Slc26a6 has affinity for oxalate, sulfate, and HCO(3)(-) in addition to Cl(-) and formate and can function in multiple exchange modes involving pairs of these anions. In the presence of high oxalate concentrations as found in renal tubular fluid and urine, Slc26a6 may largely function as an electrogenic Cl(-)-oxalate exchanger.

Ion exchangers mediating NaCl transport in the renal proximal tubule.

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. Studies of isolated brush-border membrane vesicles confirmed the presence of Na+-H+ exchange and identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanisms of uphill Cl- entry. We found that formate and oxalate each stimulate NaCl absorption in microperfused proximal tubules. Stimulation of NaCl absorption by formate was blocked by the Na+-H+-exchange inhibitor EIPA, whereas stimulation by oxalate was blocked by omission of sulfate from the perfusion solutions. These observations were consistent with recycling of formate from lumen to cell by H+-coupled formate transport in parallel with Na+-H+ exchange and recycling of oxalate by oxalate-sulfate exchange in parallel with Na+-sulfate cotransport. Using isoform-specific antibodies, we found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the loop of Henle. The inhibitor sensitivity of Na+-H+ exchange in renal brush-border vesicles and of HCO3- absorption in microperfused tubules suggested that NHE3 is responsible for most, if not all, apical membrane Na+-H+ exchange in the proximal tubule. The role of NHE3 in mediating proximal tubule HCO3- absorption and formate-dependent Cl- absorption was confirmed by studies in NHE3 null mice. Finally, we cloned and functionally expressed CFEX, an anion transporter expressed on the apical surface of proximal tubule cells and capable of mediating Cl(-)-formate exchange.

Reduction of oxaluria after an oral course of lactic acid bacteria at high concentration

Hyperoxaluria is a major risk factor for renal stones, and in most cases, it appears to be sustained by increased dietary load or increased intestinal absorption. Previous studies have shown that components of the endogenous digestive microflora, in particular Oxalobacter formigenes, utilize oxalate in the gut, thus limiting its absorption. We tested the hypothesis of whether oxaluria can be reduced by means of reducing intestinal absorption through feeding a mixture of freeze-dried lactic acid bacteria. Six patients with idiopathic calcium-oxalate urolithiasis and mild hyperoxaluria (>40 mg/24 h) received daily a mixture containing 8 x 10(11) freeze-dried lactic acid bacteria (L. acidophilus, L. plantarum, L. brevis, S. thermophilus, B. infantis) for four weeks. The 24-hour urinary excretion of oxalate was determined at the end of the study period and then one month after ending the treatment. The ability of bacteria to degrade oxalate and grow in oxalate-containing media, and the gene expression of Ox1T, an enzyme that catalyzes the transmembrane exchange of oxalate, also were investigated. The treatment resulted in a great reduction of the 24-hour excretion of oxalate in all six patients enrolled. Mean levels +/- SD were 33.5 +/- 15.9 mg/24 h at the end of the study period and 28.3 +/- 14.6 mg/24 h one month after treatment was interrupted compared with baseline values of 55.5 +/- 19.6 mg/24 h (P < 0.05). The treatment was associated with a strong reduction of the fecal excretion of oxalate in the two patients tested. Two bacterial strains among those used for the treatment (L. acidophilus and S. thermophilus) proved in vitro to degrade oxalate effectively, but their growth was somewhat inhibited by oxalate. One strain (B. infantis) showed a quite good degrading activity and grew rapidly in the oxalate-containing medium. L. plantarum and L. brevis showed a modest ability to degrade oxalate even though they grew significantly in oxalate-containing medium. No strain expressed the Ox1T gene. The urinary excretion of oxalate, a major risk factor for renal stone formation and growth in patients with idiopathic calcium-oxalate urolithiasis, can be greatly reduced with treatment using a high concentration of freeze-dried lactic acid bacteria. We postulate that the biological manipulation of the endogenous digestive microflora can be a novel approach for the prevention of urinary stone formation.

Influence of Oxalate and Soil Organic Matter on Sorption and Desorption of Phosphate onto a Spodic Horizon

AbstractPhosphate (PO4) availability limits the productivity of pine plantations growing on Spodosols of the southeastern USA. Oxalate has been shown to interact with both the sorption and desorption PO4 onto soil mineral surfaces. In addition, organic matter, a crucial component of many soil surfaces, affects the adsorption of PO4. We studied the effects of oxalate and organic matter on PO4 sorption and desorption onto the whole soil and clay‐sized fraction of a spodic horizon from a poorly drained Spodosol of the flatwoods region of the lower Coastal Plain of the southeastern USA. Common batch studies and mass balance of OH‐ production and consumption were used to interpret the processes. Maximum reduction in PO4 sorption was observed in samples where organic matter and oxalate were present. The molar ratio of OH‐ ions released to PO4 sorbed supports the idea of a ligand‐exchange mechanism dominating the PO4 sorption process. Some of the sorption sites appear to be common sites for PO4, oxalate, and organic matter. Phosphorus desorption from the spodic horizon by the action of oxalate was through ligand exchange of oxalate for PO4. The presence of soil organic matter increased the amount of PO4 desorbed by oxalate. Oxalate appeared to form stable soluble complexes with Al in solution, thus inhibiting its reprecipitation.

Effect of metabolic acidosis on NaCl transport in the proximal tubule.

In metabolic acidosis, the capacity of the proximal tubule for bicarbonate absorption is enhanced, whereas NaCl reabsorption is inhibited. Recent evidence indicates that transcellular NaCl absorption in the proximal tubule is mediated by apical membrane Cl/formate exchange and Cl/oxalate exchange, in parallel with recycling of these organic anions. We evaluated whether the effect of metabolic acidosis to inhibit NaCl reabsorption in the proximal tubule is due at least in part to inhibition of organic anion-dependent NaCl transport in this nephron segment. Absorption rates of bicarbonate (JHCO3), chloride (JCl), and fluid (Jv) were measured in rat proximal tubule segments microperfused in situ. We confirmed that metabolic acidosis stimulates JHCO3 in tubules microperfused with 25 mM HCO3, pH 7.4. For measurements of JCl, tubules were microperfused with a low-bicarbonate (5 mM), high-chloride solution, simulating conditions in the late proximal tubule. Under these conditions, baseline JCl and Jv measured in the absence of formate and oxalate were not significantly different between control and acidotic rats. However, whereas addition of 50 ¿M formate or 1 ¿M oxalate to luminal and capillary perfusates markedly stimulated JCl and Jv in control rats, formate and oxalate failed to stimulate JCl and Jv in acidotic rats. We conclude that metabolic acidosis markedly downregulates organic anion-stimulated NaCl absorption, thereby allowing differential regulation of proximal tubule NaHCO3 and NaCl transport.

In situ characterization of oxalate transport across the basolateral membrane of the proximal tubule.

Oxalate transport across the contraluminal membrane of the proximal tubule was studied in vivo using the "capillary stopped flow microperfusion method" (Pflügers Arch 400:250-256, 1984). Cellular uptake of oxalate was characteristic of a carrier-mediated transport process (Jmax = 1.6 +/- 0.6 pmol/s per cm proximal tubular length, Km = 2.03 +/- 0.77 mmol/l). Sulphate inhibited oxalate transport in a dose-dependent manner (Ki-value = 1.53 +/- 0.38 mmol/l). Sulphate transport across the basolateral membrane was also characteristic of a carrier- mediated transport process (Jmax = 1.83 +/- 0.56 pmol/s per cm proximal tubular length, Km = 1.37 +/- 0.57 mmol/l). Oxalate inhibited the sulphate transport in a dose-dependent manner (Ki = 2. 06 +/- 0.82 mmol/l). No significant differences were found between the Ki values and the Km values of the two substances, indicating that oxalate and sulphate are transported by the same carrier across the basolateral membrane of the proximal tubule. Oxalate transport was not dependent on the extracellular sodium or potassium concentration. Bicarbonate competitively inhibited the oxalate transport. Chloride significantly inhibited the oxalate transport, but not dose dependently. It is, therefore, suggested that oxalate is transported into the cell of the proximal tubule in exchange for sulphate or bicarbonate. The dose-independent inhibition by chloride is suggested to be mediated by the coupling of the sulphate (bicarbonate)/oxalate exchanger with the chloride/bicarbonate exchanger at the basolateral membrane of the proximal tubule. This, furthermore, suggests that the transport of oxalate or sulphate across the basolateral membrane might be indirectly coupled with the reabsorption of chloride at this membrane side.

Enhanced Oxalate Transport in Nucleated Red Blood Cells

Objective: This study was aimed to determine the presence of oxalate exchange/binding protein in the nucleated red blood cells (RBC). Materials and Methods: Intact RBC, nuclei, RBC membrane, nuclear membrane and histones were isolated and ¹⁴C-oxalate exchange/binding was studied. Results: The rate of oxalate anion exchange by nucleated erythrocytes was found to be higher (K = 1.24/min) than that of human RBC (K = 0.51/min) but less than that of nuclei (K = 1.55/min). Oxalate flux rate was pH- and temperature-dependent. Oxalate exchange was reduced by 35% for both chicken RBC and nuclei in presence of sulfate, bicarbonate, phosphate and succinate. Both plasma membrane and nuclear membrane showed maximal oxalate binding activity at pH 7.4, while in the nuclear basic protein fraction it was at pH 4.5. 4,4′-Diisothiocyanostilbene-2,2′-disulfonate, an anion transport inhibitor, inhibited the oxalate exchange and binding activity in both erythrocytes as well as nucleus. Conclusion: Nucleated RBC can be useful model system for studying the interaction of oxalate with nuclear components.

Effects of sulfate and chloride on three separate oxalate transporters reconstituted from rabbit renal cortex.

Understanding the mechanism of sulfate-dependent, oxalate-stimulated chloride reabsorption in the mammalian proximal tubule is complicated by the presence of multiple oxalate and sulfate transport pathways. Accordingly, we developed a method of reconstituting functional oxalate transport from the rabbit renal cortex so that the individual transporters might be examined. Solubilized microvillus membrane proteins were separated by hydroxyapatite chromatography and then reconstituted into proteoliposomes. Two peaks of oxalate/oxalate exchange activity were observed. Sulfate (10 mM) cis-inhibits oxalate transport in the early peak by 93% and in the later peak by 41%. In contrast, 20 mM chloride inhibits oxalate/oxalate exchange by only 32% in the early peak but inhibits oxalate exchange by 70% in the later peak. Oxalate-stimulated sulfate uptake was observed in the early fractions but not in the later fractions. These data are consistent with the recovery of the sulfate/oxalate exchanger in the early hydroxyapatite fractions and the chloride/oxalate exchanger in the later fractions. The basolateral membrane sulfate/oxalate exchanger was also reconstituted. The reconstituted basolateral and apical membrane sulfate/oxalate exchangers demonstrate nearly identical patterns of substrate specificities. However, 98% of apical sulfate/oxalate exchange activity is lost following exposure to octylglucoside at room temperature, whereas the basolateral sulfate/oxalate exchange activity was reduced 67% (P < 0.05). In conclusion, functional reconstitution of solubilized membrane proteins demonstrates that apical membrane chloride/oxalate exchange and sulfate/oxalate exchange are mediated by different transport proteins. Apical and basolateral sulfate/oxalate exchange may also represent transport on two separate exchangers.

Mechanisms of stimulation of proximal tubule chloride transport by formate and oxalate.

We have previously demonstrated that formate and oxalate stimulate volume absorption in the rat proximal tubule, consistent with Cl-/formate and Cl-/oxalate exchange process across the apical membrane. To sustain Cl- absorption by these processes requires mechanisms for recycling formate and oxalate from lumen to cell. The aims of the present study were to characterize these mechanisms of formate and oxalate recycling. Proximal tubules and peritubular capillaries were simultaneously microperfused in the rat kidney in situ. Serum formate concentration was determined to be 56.5 +/- 7.7 microM. Addition of 5, 50, and 500 microM formate to both luminal and capillary perfusates significantly increased net Cl- absorption (Jcl) by 26, 26, and 46%, respectively. Jcl was stimulated 38% by 1 microM oxalate added to the perfusates. Removal of sulfate completely prevented the stimulation of Jcl by 1 microM oxalate but had no effect on the stimulation of Jcl by formate. Luminal addition of the Na+/H+ exchange inhibitor ethylisopropylamiloride completely blocked the stimulation of Jcl by 50 microM formate but had no effect on stimulation by oxalate. We conclude that physiological concentrations of formate and oxalate markedly stimulate Cl- and fluid absorption in the rat proximal convoluted tubule. Whereas formate recycling most likely involves Na+/H+ exchange in parallel with H(+)-coupled formate entry, oxalate recycling involves sodium-sulfate cotransport in parallel with sulfate/oxalate exchange.

Pathways for Oxalate Transport in Rabbit Renal Microvillus Membrane Vesicles

Recent evidence suggests that apical membrane Cl--oxalate exchange plays a major role in mediating Cl- absorption in the renal proximal tubule. To sustain steady-state Cl- absorption by a mechanism of exchange for intracellular oxalate requires the presence of one or more pathways for recycling oxalate from lumen to cell. Accordingly, we evaluated the mechanisms of oxalate transport in luminal membrane vesicles isolated from the rabbit renal cortex. We found that transport of oxalate by Na+ cotransport is negligible compared to the transport of sulfate. In contrast, we demonstrated that oxalate shares the electroneutral pathway mediating Na+-independent sulfate-carbonate exchange. We also demonstrated the presence of OH--oxalate exchange (indistinguishable from H+-oxalate cotransport). The process of OH--oxalate exchange was electrogenic and partially inhibited by Cl-, indicating that it occurs, at least in part, as a mode of the Cl--oxalate exchanger described previously. An additional component of OH--oxalate exchange was insensitive to inhibition by either Cl- or sulfate, suggesting that it takes place by neither the Cl--oxalate exchanger nor the sulfate-carbonate exchanger. We conclude that multiple anion exchange mechanisms exist by which oxalate can recycle from lumen to cell to sustain Cl- absorption occurring via apical membrane Cl--oxalate exchange in the renal proximal tubule.

Role of ion exchangers in mediating NaCl transport in the proximal tubule

The reabsorption of NaCl in the proximal tubule occurs passively through the paracellular pathway, and actively by a transcellular route. Transcellular NaCl transport involves Na(+)-coupled Cl- entry across the apical membrane by two mechanisms involving Cl(-)-organic anion exchange. One mechanism is Cl(-)-formate exchange with recycling of formate from lumen to cell by H(+)-coupled formate transport in parallel with Na(+)-H+ exchange. A second mechanism is Cl(-)-oxalate exchange with recycling of oxalate from lumen to cell by oxalate-sulfate exchange in parallel with Na(+)-sulfate cotransport. Cl- exit across the basolateral membrane is most likely mediated by Cl- channels. Apical membrane Na(+)-H+ exchange is involved in mediating both NaHCO3 and NaCl reabsorption in the proximal tubule. Immunocytochemical studies indicate that NHE3 is the principal Na(+)-H+ exchanger isoform expressed on the brush border membrane. Detection of NHE3 in a subapical, intracellular, vesicular compartment in proximal tubule cells is consistent with its possible regulation by membrane trafficking. That NHE3 is the isoform responsible for apical membrane Na(+)-H+ exchange activity is supported by studies of inhibitor sensitivity, and by studies demonstrating increased expression of NHE3 protein in association with enhanced Na(+)-H+ exchange activity during renal maturation and in response to glucocorticoids and metabolic acidosis.

Anomalous phospholipid n-6 polyunsaturated fatty acid composition in idiopathic calcium nephrolithiasis.

Anomalies in the erythrocyte transport of anions and cations have been described in idiopathic calcium oxalate nephrolithiasis and seem to play a pathogenetic role in this disease. In consideration of the hypothesis that the complex array of ion flux cell abnormalities is an epiphenomenon of an anomaly in the composition of cell membranes, this study investigated cell-membrane lipid composition. In idiopathic calcium oxalate renal stone formers, in which ion transport abnormalities were present, and in healthy control subjects, plasma and erythrocyte membrane lipid composition, the erythrocyte oxalate exchange, and Na/K/2Cl cotransport activity were evaluated. Furthermore, in stone formers, the effect of a 30-day fish-oil diet supplementation on plasma lipids, erythrocyte oxalate exchange, oxaluria, and calciuria was investigated. The effect of archidonic acid released by phospholipase A2 on anion-carrier phosphorylation and activity in erythrocytes was evaluated as well. Patients had a lower content of linoleic and higher concentration of archidonic acids in both plasma and erythrocyte membrane phospholipids, and an increased archidonic/linoleic acid ratio. The archidonic acid level correlated with the erythrocyte oxalate exchange and sodium cotransport activity. Fish-oil supplementation lowered calcium and oxalate urine excretion, and normalized the erythrocyte oxalate exchange. Phospholipase A2 increased the erythrocyte anion-carrier protein phosphorylation and the oxalate exchange. This study shows that idiopathic calcium nephrolithiasis in the patient group reported here is characterized by a systemic defect in phospholipid archidonic acid levels that might provide an answer to the link between genetic background, dietary habits, and renal lithiasis.

Cloning, Sequencing, and Expression in Escherichia coli of OxlT, the Oxalate:Formate Exchange Protein of Oxalobacter formigenes

OxlT is the oxalate/formate exchange protein that represents the vectorial component of a proton-motive metabolic cycle in Oxalobacter formigenes. Here we report the cloning and sequencing of OxlT and describe its expression in Escherichia coli. The OxlT amino acid sequence specifies a polytopic hydrophobic protein of 418 residues with a mass of 44,128 daltons. Analysis of hydropathy and consideration of the distribution of charged residues suggests an OxlT secondary structure having 12 transmembrane segments, oriented so that the N and C termini face the cytoplasm. Expression of OxlT in E. coli coincides with appearance of a capacity to carry out the self-exchange of oxalate and the heterologous, electrogenic exchange of oxalate with formate. The unusually high velocity of OxlT-mediated transport is also preserved in E. coli. We conclude that the essential features of OxlT are retained on its expression in E. coli.

Characterization of oxalate transport by the human erythrocyte band 3 protein.

This paper describes characteristics of the transport of oxalate across the human erythrocyte membrane. Treatment of cells with low concentrations of H2DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) inhibits Cl(-)-Cl- and oxalate-oxalate exchange to the same extent, suggesting that band 3 is the major transport pathway for oxalate. The kinetics of oxalate and Cl- self-exchange fluxes indicate that the two ions compete for a common transport site; the apparent Cl- affinity is two to three times higher than that of oxalate. The net exchange of oxalate for Cl-, in either direction, is accompanied by a flux of H+ with oxalate, as is also true of net Cl(-)-SO4(2-) exchange. The transport of oxalate, however, is much faster than that of SO4(2-) or other divalent anions. Oxalate influx into Cl(-)-containing cells has an extracellular pH optimum of approximately 5.5 at 0 degrees C. At extracellular pH below 5.5 (neutral intracellular pH), net Cl(-)-oxalate exchange is nearly as fast as Cl(-)-Cl- exchange. The rapid Cl(-)-oxalate exchange at acid extracellular pH is not likely to be a consequence of Cl- exchange for monovalent oxalate (HOOC-COO-; pKa = 4.2) because monocarboxylates of similar structure exchange for Cl- much more slowly than does oxalate. The activation energy of Cl(-)-oxalate exchange is about 35 kCal/mol at temperatures between 0 and 15 degrees C; the rapid oxalate influx is therefore not a consequence of a low activation energy. The protein phosphatase inhibitor okadaic acid has no detectable effect on oxalate self-exchange, in contrast to a recent finding in another laboratory (Baggio, B., L. Bordin, G. Clari, G. Gambaro, and V. Moret. 1993. Biochim. Biophys. Acta. 1148:157-160.); our data provide no evidence for physiological regulation of anion exchange in red cells.

Active uptake of oxalate in a renal tubular cell line (LLC-PK1)

The oxalate uptake was studied in LLC-PK1 cells, an epithelial cell line originated in proximal tubular cells of porcine kidney. It was clear that the cells contain an oxalate exchanger highly sensitive to 4,4-diisothio-cyanostilbene-2-' disulfonic acid (DIDS). The uptake was inhibited by the addition of inorganic anions (chloride, sulphate and bicarbonate) to the reaction system, but was unaffected by sodium ion. The data suggest the possibility that it should be consistent with at least two transport systems for oxalate in LLC-PK1 cells, a SO4-/oxalate/HCO3- exchanger and a Cl-/oxalate exchanger.

Measurement of the substrate dissociation constant of a solubilized membrane carrier. Substrate stabilization of OxlT, the anion exchange protein of Oxalobacter formigenes.

OxlT, a secondary carrier found in Oxalobacter formigenes, mediates the exchange of divalent oxalate and monovalent formate. Because OxlT has an unusually high turnover number (greater than or equal to 1000/s), and because formate, one its substrates, shows high passive membrane permeability as formic acid, it has been difficult to obtain information on protein-substrate interactions using traditional methods in membrane biology. For this reason, we devised a new way to measure substrate dissociation constants. Detergent-solubilized material was exposed to inactivating temperatures in the absence or presence of OxlT substrates, and periodic reconstitution was used to monitor the kinetics of thermal decay. The data were consistent with a simple scheme in which only unliganded OxlT was temperature-sensitive; this premise, along with the assumption of equilibrium between liganded and unliganded species, allowed calculation of substrate dissociation constants for oxalate (18 +/- 3 microM), malonate (1.2 +/- 0.2 mM), and formate (3.1 +/- 0.6 mM). Further analysis revealed that substrate binding energy contributed at least 3.5 kcal/mol to stabilization of solubilized OxlT. Accordingly, we suggest that substrate binding energy is directly involved in driving protein structure reorganization during membrane transport. This new approach to analyzing protein-substrate interactions may have wider application in the study of membrane carriers.

Identification, purification, and reconstitution of OxlT, the oxalate: formate antiport protein of Oxalobacter formigenes.

We had proposed earlier that the anaerobe Oxalobacter formigenes sustains a proton-motive force by exploiting a secondary carrier rather than a primary proton pump. In this view, a carrier protein would catalyze the exchange of extracellular oxalate, a divalent anion, and intracellular formate, the monovalent product of oxalate decarboxylation. Such an electrogenic exchange develops an internally negative membrane potential, and since the decarboxylation reaction consumes an internal proton, the combined activity of the carrier and the soluble decarboxylase would constitute an "indirect" proton pump with a stoichiometry of 1H+ per turnover. This model is now verified by identification and purification of OxlT, the protein responsible for the anion exchange reaction. Membranes of O. formigenes were solubilized at pH 7 with 1.25% octyl glucoside in 20 mM 3-(N-morpholino)propanesulfonic acid/K, in the presence of 0.4% Escherichia coli phospholipids and with 20% glucerol present as the osmolyte stabilant. Rapid methods for reconstitution were developed to monitor the distribution of OxlT during biochemical fractionation, allowing its purification by sequential anion and cation exchange chromatography. OxlT proved to be a single hydrophobic polypeptide, of 38 kDa mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a turnover number estimated as at least 1000/s. The properties of OxlT point to an indirect proton pump as the mechanism by which a proton-motive force arises in O. formigenes, and one may reasonably argue that indirect proton pumps take part in bacterial events such as acetogenesis, malolactate fermentation, and perhaps methanogenesis.

Sulfate and oxalate exchange for bicarbonate across the basolateral membrane of rabbit ileum

The purpose of these studies was to further define the transport of SO4(2-) and oxalate across the basolateral membrane (BLM) of rabbit ileum. We previously found evidence for Cl-(-)SO4(2-) exchange but no evidence for carrier-mediated oxalate transport. A HCO3- gradient (but not a pH gradient) was found to stimulate SO4(2-) and oxalate uptake in BLM vesicles; uptake was inhibited by DIDS. Oxalate cis-inhibited HCO3- gradient SO4(2-) uptake and trans-stimulated SO4(2-) uptake, suggesting SO4(2-) and oxalate used the same carrier (SO4(2-)- and oxalate-HCO3-exchange). Cl- had no effect on HCO3- gradient-stimulated SO4(2-) uptake, indicating that Cl(-)-SO4(2-) exchange was a different carrier. Other substrates found to be transported on the HCO3- exchanger were oxaloacetate and S2O3(2-)-.SO4(2-), S2O3(2-). and oxalate were found to also use the SO4(2-)-Cl- exchanger, whereas oxalate was only transported on the HCO3- exchanger. SO4(2-)-HCO3- exchange was found on villus, but not crypt cell, BLM. These results indicate that there is a SO4(2-)-HCO3- exchanger on the BLM of villus cells that also transports oxalate, and along with our previous studies, the results provide evidence for the transepithelial transport of oxalate.

Oxalate:formate exchange: The basis for energy coupling in Oxalobacter

In the Gram-negative anaerobe, Oxalobacter formigenes, the generation of metabolic energy depends on the transport and decarboxylation of oxalate. We have now used assays of reconstitution to study the movements of oxalate and to characterize the exchange of oxalate with formate, its immediate metabolic derivative. Membranes of O. formigenes were solubilized with octyl-beta-D-glucopyranoside in the presence of 20% glycerol and Escherichia coli phospholipid, and detergent extracts were reconstituted by detergent dilution. [14C]Oxalate was taken up by proteoliposomes loaded with unlabeled oxalate, but not by similarly loaded liposomes or by proteoliposomes containing sulfate in place of oxalate. Oxalate transport did not depend on the presence of sodium or potassium, nor was it affected by valinomycin (1 microM), nigericin (1 microM), or a proton conductor, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (5 microM) when potassium was at equal concentration on either side of the membrane. Such data suggest the presence of an overall neutral oxalate self-exchange, independent of common cations or anions. Kinetic analysis of the reaction in proteoliposomes gave a Michaelis constant (Kt) for oxalate transport of 0.24 mM and a maximal velocity (Vmax) of 99 mumol/min/mg of protein. A direct exchange of oxalate and formate was indicated by the observations that formate inhibited oxalate transport and that delayed addition of formate released [14C]oxalate accumulated during oxalate exchange. Moreover, [14C]formate was taken up by oxalate-loaded proteoliposomes (but not liposomes), and this heterologous reaction could be blocked by external oxalate. Further studies, using formate-loaded proteoliposomes, suggested that the heterologous exchange was electrogenic. Thus, for assays in which N-methylglucamine served as both internal and external cation, formate-loaded particles took up oxalate at a rate of 2.4 mumol/min/mg of protein. When external or internal N-methylglucamine was replaced by potassium in the presence of valinomycin, there was, respectively, a 7-fold stimulation or an 8-fold inhibition of oxalate accumulation, demonstrating that net negative charge moved in parallel with oxalate during the heterologous exchange. The work summarized here suggests the presence of an unusually rapid and electrogenic oxalate2-:formate1- antiport in membranes of O. formigenes. Since a proton is consumed during the intracellular decarboxylation that converts oxalate into formate plus CO2, antiport of oxalate and formate would play a central role in a biochemical cycle consisting of (a) oxalate influx, (b) oxalate decarboxylation, and (c) formate efflux.(ABSTRACT TRUNCATED AT 400 WORDS)