Sulfate Affinity Research Articles

Isolation and purification of anaphylactic antibody from guinea pig and preliminary research on its role in passive cutaneous anaphylaxis test (PCA)

Objective To establish guinea pig model of type Ⅰ anaphylaxis, isolate the anaphy-lactic antibody(IgE and IgG) preliminarily from sera of sensitized guinea pigs, and investigate its functional characteristics. Methods Animal model was established by sensitizing guinea pigs with OVA and Al(OH)_3, level of antibody was determined by ELISA, IgE and IgG in sera were preliminarily isolated through saturated ammonium sulphate precipitate and affinity chromatograph of Protein A. A continuous passive cutaneous ana-phylaxis test (PCA) was performed by sensitizing guinea pigs individually with IgE and IgG and then challenging at different time , and the variation of blue spots in skin were observed after challenge . Results Concentration of IgE in model group and control group were 719.3750 ng/ml and 2.5250 ng/ml, the optical density of IgG in model group and control group were 0.9921 and 0.0174, the level of two antibodies in model group were significantly higher than that of control group (P<0.05). In 9 d continuous PCA test, the blue spot induced by IgE in skin lasted for 9 days and appeared the largest when challenged at day 5. The diameter of blue spot induced by IgG was the largest when challenged at day 2 and then decreased fast. Con-clusion Anaphylactic antibodies were successfully preliminarily isolated from sera of sensitizing guinea pig, both IgE and IgG play roles in type Ⅰ anaphylaxis of guinea pig, the hypersensitive reaction induced by IgG is fast and short than that induced by IgE, and IgG may become an important surrogate marker in immunotoxic-ity evaluation(type Ⅰ anaphylaxis)of vaccine. Key words: Guinea pig; Type Ⅰ anaphylaxis; Passive cutaneous anaphylaxis; IgE; IgG

Binding of Low-Molecular-Weight Cationic Ligands to Chondroitin Sulfate as Studied by Capillary Electrophoresis Frontal Analysis

The feasibility of capillary electrophoresis frontal analysis for the study of low-molecular-weight ligand- chondroitin sulfate interactions was investigated. The interaction of the 7 cationic ligands (lidocaine, propranolol, scopolamine, N-methyl scopolamine, N-butyl scopolamine, 2-propylisochinolinium, and methyl viologen) with the glycosaminoglycan chondroitin sulfate was investigated in 0.067 M phosphate buffer, pH 7.4, at 25 o C. A frontal analysis method with a short analysis time, suitable for ranking ligands according to degree of complexation for chondroitin sulfate was developed. The double charged ligand methyl viologen possessed the highest affinity for chondroitin sulfate. Linear binding isotherms were obtained and apparent association constants were determined. Capillary electrophoresis frontal analysis may be an attractive tool for characterization of ligand-glycosaminoglycan interactions.

Passage of Dengue Virus Type 4 Vaccine Candidates in Fetal Rhesus Lung Cells Selects Heparin-Sensitive Variants That Result in Loss of Infectivity and Immunogenicity in Rhesus Macaques

Three dengue virus type 4 (DENV-4) vaccine candidates containing deletions in the 3' noncoding region were prepared by passage in DBS-FRhL-2 (FRhL) cells. Unexpectedly, these vaccine candidates and parental DENV-4 similarly passaged in the same cells failed to elicit either viremia or a virus-neutralizing antibody response. Consensus sequence analysis revealed that each of the three viruses, as well as the parental DENV-4 when passaged in FRhL cells, rapidly acquired a single Glu327-Gly substitution in domain III (DIII) of the envelope protein (E). These variants appear to have accumulated in response to growth adaptation to FRhL cells as shown by growth analysis, and the mutation was not detected in the virus following passage in C6/36 cells, primary African green monkey kidney cells, or Vero cells. The Glu327-Gly substitution was predicted by molecular modeling to increase the net positive charge on the surface of E. The Glu(327)-Gly variant of the full-length DENV-4 selected after three passages in FRhL cells showed increased affinity for heparan sulfate compared to the unpassaged DENV-4, as measured by heparin binding and infectivity inhibition assays. Evidence indicates that the Glu327-Gly mutation in DIII of the DENV-4 E protein was responsible for reduced infectivity and immunogenicity in rhesus monkeys. Our results point out the importance of cell substrates for vaccine preparation since the virus may change during passages in certain cells through adaptive selection, and such mutations may affect cell tropism, virulence, and vaccine efficacy.

Effect of Environmental Conditions on Sulfate Reduction with Methane as Electron Donor by an Eckernförde Bay Enrichment

Sulfate reduction (SR) coupled to anaerobic oxidation of methane (AOM) is meditated by marine microorganisms and forms an important process in the global sulfur and carbon cycle. In this research, the possibility to use this process for the removal and recovery of sulfur and metal compounds from waste streams was investigated. A membrane bioreactor was used to enrich for a community of methane-oxidizing sulfate-reducing microorganisms from Eckernförde Bay sediment The AOM and SR rate of the obtained enrichment were 1.0 mmol gvss(-1) d(-1). The operational window and optimal environmental conditions for SR with methane as electron donor were assessed. The optimum pH, salinity, and temperature were 7.5, 30% per hundred and 20 degrees C, respectively. The enrichment had a good affinity for sulfate (Km < 0.5 mM) and a low affinity for methane (Kn > 0.075 MPa). A0M coupled to SR was completely inhibited at 2.4 (L0.1) mM sulfide. AOM occurred with sulfate, thiosulfate, and sulfite as electron accepters. Sulfate reduction with methane as electron donor can be applied for the removal of sulfate or for the production of sulfide,for metal precipitation. However, the low optimal temperature and the high salt requirement limit the operational window of the process.

Mass spectrometric study of oligourea macrocycles and their anion binding behavior

Two series, one of tris-urea macrocycles and another of hexakis-urea macrocycles, are examined by (tandem) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry with respect to their fragmentation patterns and anion binding properties. All macrocycles are based on two different building blocks, one of which is a very rigid xanthene unit and the other one is a more flexible diphenyl ether. The composition and the sequence of these units thus determine their flexibility. During the fragmentation of deprotonated oligourea macrocycles in the gas phase, one urea N-CO bond is cleaved followed by a scrambling reaction within the macrocycle structure. Consequently, fragments are observed that deviate from those that would be expected from the sequence of the subunits. Interesting anion binding properties involve the simultaneous recognition of two chloride anions by one of the hexakis-urea macrocycles, whose flexibility allows this host to form a double-helical structure. Flexibility also determines which of the hexameric receptors bears a high sulfate affinity. The interaction energy between some of the macrocycles and sulfate is high enough to even stabilize the intrinsically unstable sulfate dianion.

A novel and one-step purification of human ceruloplasmin by acharan sulfate affinity chromatography

Human ceruloplasmin, a copper binding alpha(2)-glycoprotein, was purified by a single-step procedure using acharan sulfate affinity chromatography. Acharan sulfate was immobilized to amine-functionalized agarose matrix through carboxylic acids. Ceruloplasmin in human plasma was obtained from 0.4 M NaCl salt elution and characterized by SDS-PAGE (132 and 125 kDa), isoelectric focusing (pI 4.6), Western blotting, and MALDI-TOF-MS peptide mass fingerprinting. Ceruloplasmin was purified 106 fold with a specific oxidase activity of 0.53 U/mg protein.

The role of heparan sulfate on adhesion of 47 and 51 kDa outer membrane proteins ofHelicobacter pylorito gastric cancer cellsThis study was done in memory of the late Dr. Roberto Carlos Vazquez-Juarez.

Helicobacter pylori is a common gastrointestinal pathogenic bacterium in humans and the usual preference for the stomach's outer membrane proteins (OMPs) are antigens involved in the adhesion process. Through SDS-PAGE and blotting analyses, using horseradish peroxidase-labeled heparan sulfate (HRP-HS) as a probe, we identified H. pylori OMPs with affinity for heparan sulfate (OMP-HS). Biotin-streptavidin bacterial-adhesion assay was used to evaluate participation of OMP-HS in the adhesion of H. pylori to semi-confluent HeLa S3 and Kato III cell monolayers. The results provide evidence that induction of antibodies against 2 OMP-HSs (HSBP-47 and HSBP-51) could reduce binding of H. pylori to both cell lines and induce detachment of cell-bound bacteria from infected cultured cells.

A Mouse-Passaged Dengue Virus Strain with Reduced Affinity for Heparan Sulfate Causes Severe Disease in Mice by Establishing Increased Systemic Viral Loads

The four serotypes of dengue virus (DENV1 to DENV4) cause extensive morbidity and mortality. A major obstacle to studying disease pathogenesis and developing therapies has been the lack of a small-animal model. We previously reported isolation of a DENV2 strain, obtained by passaging a clinical isolate between mosquito cells and mice, that caused severe DENV disease in mice and contained multiple mutations, including many in domain II of the envelope (E) protein. Here, we describe a recombinant virus, differing from the non-mouse-passaged virus by two mutations in the E protein, that induces vascular leakage and tumor necrosis factor alpha (TNF-alpha)-mediated lethality, while the non-mouse-passaged virus causes paralysis. This recombinant virus has a weaker affinity for heparan sulfate, resulting in an increased serum half-life, higher systemic viral loads, and high levels of TNF-alpha in the serum of infected mice. These results exemplify the role of the E protein in modulating virion clearance and connect the effect of clearance on the systemic viral loads responsible for severe disease manifestations.

Metal–organic anion receptors: trans-functionalised platinum complexes

The anion complexation properties of a trans-functionalised platinum(II) complex have been studied revealing a high affinity for sulfate in solution and 3:2 receptor sulfate complex formation in the solid state with the anion bound in a pocket lined with 6 NH and 8 CH hydrogen-bond-donating groups.

The Characteristic High Sulfate Content in Brassica oleracea is Controlled by the Expression and Activity of Sulfate Transporters

The uptake and distribution of sulfate in BRASSICA OLERACEA, a species characterised by its high sulfate content in root and shoot, are coordinated and adjusted to the sulfur requirement for growth, even at external sulfate concentrations close to the K (m) value of the high-affinity sulfate transporters. Plants were able to grow normally and maintain a high sulfur content when grown at 5 or 10 microM sulfate in the root environment. Abundance of mRNAs for the high affinity sulfate transporters, BolSultr1;1 and BolSultr1;2, were enhanced at <or= 25 microM sulfate, and this was accompanied with an up to three-fold increase in the sulfate uptake capacity, whereas sulfate, organic sulfur, and thiol contents were only slightly affected. Upon sulfate deprivation, there was a much greater induction of the sulfate transporters, BolSultr1;1, BolSultr1;2, BolSultr1;3, BolSultr2;1, and BolSultr4;1, whilst the sulfate uptake capacity was only increased up to four-fold. Plant growth and shoot to root biomass allocation were affected only upon sulfate-deprivation and not at low external sulfate concentrations. From the current results it is suggested that the internal sulfate concentration may act as a determining factor in the regulation of activity and expression of sulfate transporters, and of shoot to root biomass allocation in B. OLERACEA.

The Vascular Wall and the Haemostatic Balance in Type 1 Diabetic Patients with Nephropathy

In type 1 diabetes a main burden of complications is related to presence of nephropathy. A generalised vascular damage in patients with nephropathy has been proposed to explain this relation. A procoagulant state in type 1 diabetes patients can be related to the transcapillary escape rate of albumin in patients without nephropathy. There is however to date, no consistently demonstrated further procoagulant derangement in patients with nephropathy, but this may be due to a methodological shortcoming caused by urinary loss of the coagulation marker measured. A significant heparin-induced vitronectin release in patients with nephropathy can reflect procoagulant activity in these patients. The procoagulant state in normoalbuminuric type 1 diabetes is followed by an increased activity of tissue- type plasminogen activator, and this potential compensation of the procoagulant state is absent in nephropathy. Haemostatic balance could be achieved at a higher level of fibrin accumulation or a higher activity of a fibrin surface exposed to the circulation. Data from a venous occlusion study indicate that plasminogen activator inhibitor 1 (PAI 1), fibronectin and von Willebrand factor were significantly captured by the subendothelial matrix in patients with nephropathy. Antithrombin with specific affinity for heparan sulphate was not significantly captured and this could be a consequence of a deficiency of heparan sulphate in the subendothelial matrix. Heparin is able to reduce urinary albumin excretion in patients with incipient nephropathy, and although the mechanism for this effect is unknown, it is theoretically in accordance with the hypothesis that heparan sulphate is a key in the development of diabetic nephropathy. Future long-term studies, perhaps with oral heparins, should be performed to investigate whether such treatment, added to the current treatment set in guidelines, could further protect patients from progression in diabetic nephropathy. Such studies could also address whether generalised vascular damage can be modified.

Sperm surface arylsulfatase A can disperse the cumulus matrix of cumulus oocyte complexes

Cumulus cell layers of expanded cumulus oocyte complexes (COCs) are interlinked with networks of hyaluronic acid, chondroitin sulfate B proteoglycans and link proteins, and they can be dispersed by sperm surface hyaluronidases. In this report, we showed that arylsulfatase A (AS-A), existing on the sperm head surface, also had this dispersion action. Purified AS-A free of protease, hyaluronidase and chondroitinase activities could disperse the cumulus matrix of expanded COCs. However, this COC dispersion action was not associated with AS-A desulfation activity, assayed by using p-nitrocatecholsulfate (artificial substrate). COCs incubated for 1 h with sperm pretreated with anti-AS-A IgG in the presence of apigenin (a hyaluronidase inhibitor) did not exhibit matrix dispersion, whereas several cumulus layers were already dispersed in COCs incubated with sperm pretreated with preimmune IgG. Furthermore, sperm from AS-A null mice showed a significant delay in COC dispersion, compared with wild-type sperm. Within 1 h of sperm-COC co-incubation, the size of COCs incubated with AS-A null sperm was 65% of the original dimension, whereas that of COCs inseminated with wild-type sperm was only 17%. A further delay in COC dispersion by AS-A(-/-) mouse sperm was observed when apigenin was present in the co-incubation. We also showed for the first time that AS-A had a specific affinity for chondroitin sulfate B, a component of cumulus matrix proteoglycan networks; this might provide a mechanism of cumulus matrix destabilization induced by sperm surface AS-A.

Isoform-specific Heparan Sulfate Binding within the Amino-terminal Noncollagenous Domain of Collagen α1(XI)

Collagen type XI is a constituent of the pericellular matrix of chondrocytes and plays a role in the regulation of fibrillogenesis. The amino-terminal domain of collagen type XI alpha1 chain is a noncollagenous structure that has been identified on the surface of cartilage collagen fibrils. The biochemical composition of the amino-terminal domain varies due to alternative splicing of the primary transcript. Recombinantly expressed alpha1(XI) aminoterminal domain isoforms were used in this study to investigate potential interactions. Purified products were analyzed for heparan sulfate binding properties. The results demonstrated that two additional binding sites exist within the alpha1(XI) aminoterminal domain, one within the amino propeptide and one within the variable region of the amino-terminal domain. Analysis of relative affinities indicated that the site located within the amino propeptide (site 1) was of similar affinity to sites that exist within the major triple helix of collagen type XI. Substitution of amino acid residues 147 to 152 within the amino propeptide by site-directed mutagenesis resulted in altered affinity for heparan sulfate. The binding site located within the variable region (site 2) demonstrated significantly higher affinity than other sites within the molecule. Displacement of collagen type XI within the pericellular matrix was observed in cell culture in the presence of excess heparan sulfate and by treatment with heparinase. These studies suggest two additional binding sites located within the noncollagenous amino-terminal domain that may play a role in the function of collagen type XI. The localization of collagen type XI within the pericellular matrix may be dependent upon interactions with heparan sulfate proteoglycans, and these are likely to take place in an isoform-specific manner.

A surface complexation model for sulfate and selenate on iron oxides consistent with spectroscopic and theoretical molecular evidence

Sulfate and selenate adsorption on iron oxides are important reactions in natural systems under a very wide range of pH values, ionic strengths, and electrolyte compositions. Under such conditions, spectroscopic and theoretical calculations have demonstrated the potential importance of a variety of surface species. Understanding the variations in the surface speciation of these oxyanions is fundamental to prediction of their partitioning between minerals and aqueous solutions. In the present study, published experimental spectroscopic and theoretical molecular evidence of the identity of sulfate/selenate surface species are integrated with a surface complexation model consistent with a wide variety of experimental adsorption, surface titration, and proton coadsorption data to define the surface speciation of sulfate and selenate on iron oxides under a wide range of conditions. The analysis was carried out with the extended triple layer model (ETLM) taking into account the electrostatics of water dipole desorption during ligand exchange reactions. On seven out of eight goethites studied, sulfate and selenate surface reactions can be represented by the formation of a monodentate-mononuclear inner-sphere and a bidentate-binuclear outer-sphere (or H-bonded) species according to > FeOH + H + + AO 4 2 - = > FeOAO 3 - + H 2 O and 2 > FeOH + 2 H + + AO 4 2 - = ( > FeOH 2 + ) 2 _ AO 4 2 - respectively, where A stands for S or Se. The model predicted changes in the proportions of the species with pH, ionic strength and surface coverage consistent with independently derived experimental evidence from in situ Raman, ATR-FTIR and EXAFS studies. In contrast to goethite, the ETLM analysis of sulfate and selenate adsorption on hydrous ferric oxide (HFO) required an additional outer-sphere (or H-bonded) surface species represented by, > FeOH + 2 H + + AO 4 2 - = > FeOH 2 + _ HAO 4 - Equilibrium constants for sulfate and selenate adsorption based on site-occupancy standard states ( K θ ) for > FeOAO 3 - and ( > FeOH 2 + ) 2 _ AO 4 2 - on HFO are systematically higher than those on goethite, indicating that HFO has a greater affinity for sulfate and selenate than goethite.

Behavior of complexing ligands during coagulation–flocculation by ferric chloride: A comparative study between sewage water and an engineered colloidal model

In this work, a comparative study is conducted on sewage as an example of a natural water system and on an engineered colloidal model. The two water systems were treated by coagulation–flocculation with ferric chloride. Affinity of phosphate and sulfate used as complexing ligands to coagulant species was studied within the two systems. Aggregation dynamics were followed with jar tests and laser diffraction. Sulfate and phosphate were determined by using ion chromatography. Atomic absorption spectroscopy (AAS) and transmission electron microscopy coupled with energy dispersive X-ray spectrometry were used for elemental and ionic analysis of P, Fe, S, SO4, and PO4 in supernatants and sediments. Investigation of ligands revealed that phosphate and iron(III) species make strong complexes that enhance aggregation velocity, whereas sulfate is weakly complexed with iron(III) species. It can be released in suspensions without any consequences on aggregation dynamics.Key words: aggregation, coagulant species, complexing ligands, coagulation, flocculation.

Recoverin Binds Exclusively to an Amphipathic Peptide at the N Terminus of Rhodopsin Kinase, Inhibiting Rhodopsin Phosphorylation without Affecting Catalytic Activity of the Kinase

Recoverin is a calcium-dependent inhibitor of rhodopsin kinase. It prevents premature phosphorylation of rhodopsin until the opening of cGMP-gated ion channels causes a decrease in intracellular calcium levels, signaling completion of the light response. This calcium depletion causes release of recoverin from rhodopsin kinase, freeing the kinase to phosphorylate rhodopsin and to terminate the light response. Previous studies have shown that recoverin is able to bind to a region at the N terminus of rhodopsin kinase. In this study we map this interaction interface, showing that residues 1-15 of the kinase form the interaction site for recoverin binding. Mutation of hydrophobic residues in this region have the greatest effect on the interaction. The periodic nature of these residues suggests that they lie along one face of an amphipathic helix. We show that this region is essential for recoverin binding, as a catalytically active kinase lacking these residues is unable to bind recoverin. In addition, we show that neither the N-terminal deletion nor the presence of recoverin inhibits the overall catalytic activity of the kinase, as measured by light-independent autophosphorylation. Finally, we observe that a kinase mutant lacking the N-terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin. Taken together, these data support a model in which recoverin prevents rhodopsin phosphorylation by sterically blocking a region of kinase essential for its interaction with rhodopsin, thereby preventing recognition of rhodopsin as a kinase substrate.

Colorimetric heparinase assay for alternative anti-metastatic activity

Heparanase has been previously associated with the metastatic potential, inflammation, and angiogenesis of tumor cells. Heparanase activity has been detected by means of UV absorption, radiolabeled substrates, electrophoretic migration, and heparan sulfate affinity assays. However, those methods have proven to be somewhat problematic with regards to application to actual biological samples, the accessibility of the immobilized substrates, experimental sensitivity, and the separation of degraded products. Rather than focusing on heparanase activity, then, we have developed a rapid, alternative colorimetric heparinase assay, on the basis of the recent finding that sulfated disaccharides generated from heparin by bacterial heparinase exhibit biological properties comparable to those from heparan sulfate by mammalian heparanase. In this study, the concentrations of porcine heparin and bacterial heparinase I were determined using a Sigma Diagnostics Kit. Morus alba was selected as a candidate through this assay system, and an inhibitor, resveratrol, was purified from its methanol extract. Its anti-metastatic effects on the pulmonary metastasis of murine B16 melanoma cells were also evaluated. Our findings suggest that this assay may prove useful as a diagnostic tool for heparinase inhibition, as an alternative anti-metastatic target.

Heavy Metal Stress and Sulfate Uptake in Maize Roots

ZmST1;1, a putative high-affinity sulfate transporter gene expressed in maize (Zea mays) roots, was functionally characterized and its expression patterns were analyzed in roots of plants exposed to different heavy metals (Cd, Zn, and Cu) interfering with thiol metabolism. The ZmST1;1 cDNA was expressed in the yeast (Saccharomyces cerevisiae) sulfate transporter mutant CP154-7A. Kinetic analysis of sulfate uptake isotherm, determined on complemented yeast cells, revealed that ZmST1;1 has a high affinity for sulfate (Km value of 14.6 +/- 0.4 microm). Cd, Zn, and Cu exposure increased both ZmST1;1 expression and root sulfate uptake capacity. The metal-induced sulfate uptakes were accompanied by deep alterations in both thiol metabolism and levels of compounds such as reduced glutathione (GSH), probably involved as signals in sulfate uptake modulation. Cd and Zn exposure strongly increased the level of nonprotein thiols of the roots, indicating the induction of additional sinks for reduced sulfur, but differently affected root GSH contents that decreased or increased following Cd or Zn stress, respectively. Moreover, during Cd stress a clear relation between the ZmST1;1 mRNA abundance increment and the entity of the GSH decrement was impossible to evince. Conversely, Cu stress did not affect nonprotein thiol levels, but resulted in a deep contraction of GSH pools. Our data suggest that during heavy metal stress sulfate uptake by roots may be controlled by both GSH-dependent or -independent signaling pathways. Finally, some evidence suggesting that root sulfate availability in Cd-stressed plants may limit GSH biosynthesis and thus Cd tolerance are discussed.

Autocrine production of biologically active hepatocyte growth factor (HGF) by injured human skin

Hepatocyte growth factor (HGF) is a potent regenerative factor involved in wound healing. Previous studies have shown that mesenchymal cells produce HGF, stimulating epithelial cells in a paracrine fashion. To examine whether autocrine HGF production by keratinocytes can occur upon skin injury. A 31-year-old male patient sustained a burn affecting 80% of his total body surface area. Biopsies were taken from intact skin near the injured area, and skin keratinocytes were separated and cultured. Conditioned medium from keratinocytes was analyzed for HGF by ELISA, surface plasmon resonance (SPR), and dot blotting. Binding of HGF from conditioned medium to its receptor, c-Met, was compared with recombinant HGF by SPR. Finally, we examined the motogenic effect on mouse transformed skin epithelial cells (CCL-53.1) of HGF from conditioned medium. HGF was detected in the cultured keratinocyte medium. Similar to recombinant HGF, HGF from conditioned medium had a high affinity for dextran sulfate and albumin, and the same epitopes were engaged by the interaction of HGF with the c-Met receptor. The conditioned medium from keratinocytes obtained from the burn patient, but not medium from keratinocytes obtained from healthy volunteers, accelerated the motogenesis of CCL-53.1 cells. Unexpectedly, anti-HGF antibodies did not prevent this effect. However, anti-c-Met antibodies completely inhibited the motogenic effect. Upon injury, human skin keratinocytes might produce biologically active HGF in an autocrine fashion. This HGF might have different structural and/or biological properties from HGF produced by mesenchymal cells.

Role of GSH in Estrone Sulfate Binding and Translocation by the Multidrug Resistance Protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.