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1.
Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (Kd) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. Kd measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca2+, Mg2+, and ethylenediammonium2+ ions). Monovalent cations (e.g., Na+, K+, and Tris+) were also effective, but only at concentrations in the millimolar range. The Kd for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl2. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.  相似文献   

2.
Binding of [3H]folate to proteins in serum of pregnant women was studied in equilibrium dialysis experiments (pH 7.4, 37°C). A Scatchard analysis revealed the presence of high-affinity (Kass = 1010M?1, N = 0.4 nM folate) and low-affinity sites. The high-affinity folate binding protein (Mr ≈ 30 000–35 000) appeared in front effluent after application of serum to a DEAE-Sepharose CL-6B column equilibrated with 0.05 M imidazole buffer (pH 6.3)/ 30 mM NaCl. Low-affinity binding protein eluted from the column after a rise in NaCl concentration to 1 M was mainly similar to albumin. A minor part was, however, associated with a large molecular size (Mr > 200 000) protein, probably α2-macroglobulin.High-affinity binding which displayed positive cooperatively was saturated at folate concentrations above 10?10 M. Folate dissociation was a complex process consisting of an initial rapid phase (terminated within 48 h) followed by a slow release. At pH 3.5 dissociation became rapid and complete. Purified methotrexate had no effect on high-affinity binding, whereas N10-methylfolate (an impurity in the methotrexate preparation) acted as a potent inhibitor. Low-affinity binding was proportional to the folate concentration within the range 10?10–10?7 M. Dissociation of folate was rapid.  相似文献   

3.
Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [3H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K i=45nm) and methotrexate (K i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.  相似文献   

4.
l-[3H]Glutamate binding to synaptic plasma membranes from rat cerebral cortices was carried out at 2–4°C in 50 mM Tris-acetate buffer (pH 7.4) using a microfuge centrifugation method. Binding was increased by repeated freezing-thawing and washing in either crude or partially purified synaptic membranes. Scatchard analysis showed a single binding site (dissociation constant, KD = 697 nM; maximal binding capacity, Bmax = 7.5 pmol/mg protein) in four times distilled water washed crude synaptic membrane. After six times freezing-thawing and washing, a new high affinity site (KD1 = 26 nM, Bmax1 = 1.8 pmol/mg protein) appeared and the number of low affinity site was increased with no apparent change in affinity (KD2 = 662 nM, Bmax2 = 10.5 pmol/mg protein). l-[3H]Glutamate binding was inhibited by acidic amino acid analogues that interact with N-methyl-d-aspartate- and quisqualate-sensitive sites of glutamate receptors. Binding was marginally inhibited by kainate and l-2-amino-4-phosphonobutyrate. These results indicate that repeatedly frozen-thawed and washed synaptic plasma membrane is suitable for studying the subtypes and regulation of glutamate receptors.  相似文献   

5.
Rat placenta contains virtually no unsaturated (i.e., apo-form) folate binding protein. However, by lowering the pH of a solubilized membrane preparation of this tissue to 3.5, the endogenous bound folate was dissociated from the protein and adsorbed to charcoal. The apo-form of the folate binding protein thus obtained was purified by affinity chromatography using pteroylglutamic acid covalently coupled to Sepharose 4B. A single protein band with an apparent Mr of 36 000 was observed by SDS-polyacrylamide gel electrophoresis of the eluate from the affinity matrix. Western blot of this preparation using a rabbit antiserum raised with the affinity eluate also identified a single 36 kDa protein band. However, peptide sequencing of the N-terminal region of the proteins in the affinity eluate established that it contained two homologous proteins. Computer alignment of the first 22 N-terminal amino acids of each rat placental protein with human, bovine milk and mouse folate binding proteins showed 50–64% identical homology and 27% homology when the eight proteins were aligned together. The affinity of both rat proteins is highest for pteroylglutamic acid (Ka = 1.6 − 109 l/mol) lower for N5-methyltetrahydrofolate and substantially lower for N5-formyltetrahydrofolate. In the dose-response range studied there was no apparent affinity for methotrexate. The folate binding proteins could be released from a preparation of placental membranes using phospholipase C indicating that these proteins belong to the class of proteins anchored to the plasma membrane by a glycosyl phosphatidylinositol adduct.  相似文献   

6.
The zero-trans influx of 500 μM uridine by CHO, P388, L1210 and L929 cells was inhibited by nitrobenzylthioinosine (NBTI) in a biphasic manner; 60–70% of total uridine influx by CHO cells and about 90% of that in P388, L1210 and L929 cells was inhibited by nmolar concentrations of NBTI (ID50 = 3?10 nM) and is designated NBTI-sensitive transport. The residual transport activity, designated NBTI-resistant transport, was inhibited by NBTI only at concentrations above 1 μM (ID50 = 10?50 μM). S49 cells exhibited only NBTI-sensitive uridine transport, whereas Novikoff cells exhibited only NBTI-resistant uridine transport. In all instances NBTI-sensitive transport correlated with the presence of between 7·104 and 7·105 high-affinity NBTI binding sites/cell (Kd = 0.3?1 nM). Novikoff cells lacked such sites. The two types of nucleoside transport, NBTI-resistant and NBTI-sensitive, were indistinguishable in substrate affinity, temperature dependence, substrate specificity, inhibition by structurally unrelated substances, such as dipyridamole or papaverine, and inhibition by sulfhydryl reagents or hypoxanthine. We suggest, therefore, that a single nucleoside transporter can exist in an NBTI-sensitive and an NBTI-resistant form depending on its disposition in the plasma membrane. The sensitive form expresses a high-affinity NBTI binding site(s) which is probably made up of the substrate binding site plus a hydrophobic region which interacts with the lipophilic nitrobenzyl group of NBTI. The latter site seems to be unavailable in NBTI-resistant transporters. The proportion of NBTI-resistant and sensitive uridine transport was constant during proportion of NBTI-resistant and sensitive uridine transport was constant during progression of P388 cells through the cell cycle and independent of the growth stage of the cells in culture. There were additional differences in uridine transport between cell lines which, however, did not correlate with NBTI sensitivity and might be related to the species origin of the cells. Uridine transport in Novikoff cells was more sensitive to inhibition by dipyridamole and papaverine than that in all other cell lines tested, whereas uridine transport in CHO cells was the most sensitive to inactivation by sulfhydryl reagents.  相似文献   

7.
Folate plays a critical role in maintaining normal metabolic, energy, differentiation and growth status of all mammalian cells. The disturbances in body folate homeostasis such as intestinal malabsorption in alcoholism are well-known contributor to folate deficiency associated disorders. The study was sought to delineate the kinetic features of folate transport in intestinal absorptive epithelium that could highlight insights of malabsorption during alcoholism. We studied [3H]-folic acid transport in intestinal brush border membrane (BBM) after 3 months of ethanol administration at 1 g/kg body weight/day to rats. The results showed that the folate transport exhibited saturable kinetics and was pH, Na+, temperature, divalent cation sensitive, besides –SH group(s) was/were found important in the folate transport system to be efficiently operative. Importantly, the decreased intestinal BBM folate transport in chronic alcoholism was associated with increased K m and decreased V max during alcoholism. In addition, S–S group status of the transporter and presence of Na+ at the absorptive site seems to be perturbed during ethanol ingestion. However, H+/folate coupled transport provided the driving force for transport as pH optimum in acidic range was not altered during alcoholism. The inhibition constants of methotrexate and unlabelled folic acid revealed that the two analogues are handled differently by the folate transport system. In addition, the low activity of folate transport system during chronic ethanol exposure was associated with low RBC folate levels. Overall, these findings suggest that the deregulated folate transport kinetics might contribute to intestinal folate malabsorption in alcoholism.  相似文献   

8.
C. Barberis 《FEBS letters》1983,162(2):400-405
Characterization of specific vasopressin binding sites to rat hippocampal membranes has been assayed using tritiated lysine-vasopressin labelled on the tyrosyl residue. At 30°C specific [3H]vasopressin binding was saturable. The estimated equilibrium dissociation constant was 7.1 nM, the mean maximal binding capacity was 78 fmol/mg protein. Arginine-vasopressin has a high affinity (Kd = 2.8 nM) and dDAVP has a low affinity (Kd = 249 nM) for hippocampal synaptic membranes. (OH)AVP and Phe2Orn8VT are at least as active as AVP in inhibiting [3H]vasopressin binding. Adenylate cyclase was activated by VIP and inhibited by PIA, but not affected by lysine-vasopressin.  相似文献   

9.
Methotrexate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide react to form an activated ester of methotrexate which is a potent irreversible inhibitor of methotrexate transport in L1210 cells. In cells treated with the reagent at 37°C, inhibition was rapid (t12 < 1 min), optimal at pH 6.8, half-maximal at an inhibitor concentration of 20 nM, and complete at high levels of the reagent. Specificity was indicated by the fact that excess methotrexate added during the pretreatment step protected the transport system against inactivation. Irreversible inhibition was also observed in cells exposed to the reagent at 4°C. Inactivation in this case was qualitatively similar to the corresponding process at 37°C; it appeared rapidly, was half-maximal at 20 nM, and could be prevented by the addition of high concentrations of the substrate. The extent of the inhibition, however, reached a maximum of only 75%, even in samples containing excess or multiple additions of reagent. The latter findings suggest that at 4°C the transport protein exists in two forms, one (75% of the total) containing binding sites which are accessible to the active ester, and the other (25% of the total) with inaccessible sites. The identity of these sites is suggested to be transport proteins which have outward and inward orientations, respectively.  相似文献   

10.
A binding component with a high affinity for 5-methyltetrahydrofolate (KD = 0.11μm) is present on the external surface of L1210 cells. The amount of binder (1 pmol/mg protein) corresponds to 8 × 104 sites per cell. The participation of this component in the high-affinity 5-methyltetrahydrofolate/methotrexate transport system is supported by similarities in the KD values for 5-methyltetrahydrofolate and methotrexate binding and the Kt values of these compounds for transport. Relative affinities for other folate substrates (aminopterin, 5-formyltetrahydrofolate, and folate) and various competitive inhibitors (thiamine pyrophosphate, ADP, AMP, arsenate, and phosphate) are also similar for both the binding component and the transport system. The measured binding activity does not represent low-temperature transport of substrate into cells, since it is readily saturable with time and is eliminated by either washing the cells with buffer or by the addition of excess unlabeled substrate.  相似文献   

11.
Folate deaminase released from cells of Dictyostelium discoideum is heterogenous with respect to molecular weight and stability at 60°C. The most heat-stable component isoelectrofocuses in a broad band at approx. pH 6. The Km value of this component for folate is approx. 7 · 10?7 M and Mr approx. 40 000. The major portion if not all of the deaminase binds to immobilized concanavalin A and lentil lectin. Extracellular folate deaminase has a pH-optimum of approx. pH 6.0. This is higher than that of lysosomal enzymes, which are also glycoproteins released into the extracellular medium.  相似文献   

12.
An L1210 cell line (JT-1), which can grow in medium supplemented with 1 nM folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37 degrees C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23 +/- 0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [3H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37 degrees C remained as unmetabolized folic acid. Binding was also rapid at 0 degrees C but uptake at the plateau was only one-half the value obtained at 37 degrees C. Half-maximal saturation of the binding component (KD) occurred at a folate concentration of 0.065 nM at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (KD = 2.0 nM). 5-Methyltetrahydrofolate was also bound by this component (Ki = 13 nM at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (Ki = 45 nM) and methotrexate (Ki = 325 nM). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500 nM caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

13.
The presence of a folate binding protein of high-affinity type (affinity constant 5 · 109M–1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand3H-folate. Dissociation of3H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (M r 25 000) and a minor one (M r 100 000).  相似文献   

14.
Transport of folate compounds into Lactobacillus Casei   总被引:5,自引:0,他引:5  
Transport of folate, 5-methyl tetrahydrofolate, and amethopterin into Lactobacillus casei occurs against a concentration gradient, is pH and temperature dependent, requires glucose, exhibits saturation kinetics, is maximal when cells are harvested in late-log phase, and is repressed by excess folate in the growth medium. Km values are 0.35, 0.90, and 0.21 μm for the influx of folate, 5-methyl tetrahydrofolate, and amethopterin, respectively. Dihydrofolate, tetrahydrofolate, 5-formyl tetrahydrofolate, 5-methyl tetrahydrofolate, aminopterin, and amethopterin are inhibitors of folate influx. Countertransport of 5-methyl tetrahydrofolate is enhanced by various other folate compounds. Uptake of folate, 5-methyl tetrahydrofolate, and amethopterin is inhibited to the same degree by increasing concentrations of iodoacetate. These results indicate that a single system is responsible for transport of a variety of folate compounds into L. casei.  相似文献   

15.
The role of nucleoside transport in ischemia-reperfusion injury and arrhythmias has been well documented in various animal models using selective blockers. However, clinical application of nucleoside transport inhibitors remains to be demonstrated in humans. It is not known whether human heart has nucleoside transport similar to that of animals. The aim of this study is to pharmacologically identify the presence of nucleoside transport binding sites in the human myocardium compared to animals.Myocardial tissue was obtained from guinea pig left and right ventricle, canine left ventricle, human intraoperative right atrium and human cadaveric right atrium and right and left ventricles. Myocardial preparations were obtained from tissue samples after homogenized and a differential centrifugation.Equilibrium binding assays were performed using [3H]-p-nitrobenzylthioinosine (NBMPR) at room temperature in the presence or absence of non-radioactive NBMPR or other nucleoside transport blockers such as p-nitrobenzylthioguanosine dipyridamole, lidoflazine, papaverin, adenosine and doxorubcine. From saturation curves and inhibition kinetics, we determined the relative maximal binding (Bmax) and dissociation constant (Kd) of [3H]-NBMPR binding of human myocardial preparations.Results demonstrated that the fresh human myocardial preparations have a specific binding site for NBMPR with a Bmax of 283 ± 32 fmol/mg protein and Kd of 0.56 ± 0.12 nM. These values are lower than those obtained from guinea pigs (Bmax = 1440 ± 187 fmol/mg protein and Kd = 0.21 ± 0.03 nM) and canine atrium (Bmax 594 ± 73 fmol/mg protein, and Kd = 1.12 ± 0.22 nM).Displacement kinetics studies revealed the relative potencies (of certain unrelated drugs as follow: p-nitrobenzylthioguanosine > dipyridamole > lidoflazine > pavaverine > Diltazam > adenosine > doxyrubicin. It is concluded that human myocardium contains an active nucleoside transport site which may play a crucial role in post-ischemic reperfusion-mediated injury in a wide spectrum of ischemic syndromes.  相似文献   

16.
The presence of a folate binding protein of high-affinity type (affinity constant 3.1010M–1, maximum folate binding 1.4 nM) in human semen was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand3H-folate. Radioligand dissociation from the binding protein was slow at pH 7.4, but rapid at pH 3.5. By use of rabbit antibodies against 25 kDa human milk folate binding protein we determined the concentration of folate binding protein in 16 speciments of human semen in an enzyme-linked immunosorbent assay. The concentration of immunoreactive folate binding protein was independent of the number of spermatozoa in individual specimens. Gel filtration showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one of 100 kDa and a minor one of 25 kDa.  相似文献   

17.
C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric acid cycle, while in anaerobic bacteria, they are important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes, but only one of these utilizes an extracytoplasmic solute binding protein to achieve high-affinity uptake. Here, we identify the MatBAC system from the photosynthetic bacterium Rhodopseudomonas palustris as the first member of the tripartite tricarboxylate transport family to be involved in C4-dicarboxylate transport. Tryptophan fluorescence spectroscopy showed that MatC, the periplasmic binding protein from this system, binds to l- and d-malate with Kd values of 27 and 21 nM, respectively, the highest reported affinity to date for these C4-dicarboxylates, and to succinate (Kd = 110 nM) and fumarate (Kd = 400 nM). The 2.1-Å crystal structure of MatC with bound malate shows a high level of substrate coordination, with participation of two water molecules that bridge hydrogen bonds between the ligand proximal carboxylic group and the main chain of two conserved loops in the protein structure. The substrate coordination in MatC correlates with the binding data and explains the protein's selectivity for different substrates and respective binding affinities. Our results reveal a new function in C4-dicarboxylate transport by members of the poorly characterized tripartite tricarboxylate transport family, which are widely distributed in bacterial genomes but for which details of structure–function relationships and transport mechanisms have been lacking.  相似文献   

18.
High-affinity binding of [3H]folate in human urine displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. By means of a two-site enzyme-linked immunosorbent assay (ELISA) with rabbit antibodies against the low molecular weight folate binding protein from human milk, we measured folate binding protein concentrations in the range of 0.51 to 4.13 nM in urine samples from 16 apparently healthy individuals. Ultrogel AcA 44 chromatography of the urine showed that immunoreactive and radioligand bound folate binding protein coeluted in one large peak (Mr25,000).  相似文献   

19.
An endogenous inhibitor of γ-aminobutyric acid (GABA) receptors was partially purified from bovine brain striatum. It was obtained as a low molecular weight fraction by gel filtration on Biogel P-2 and was adsorbed to Dowex AG 50W-X8, but not to Dowex AG 1-X8. It was ninhydrin-negative, basic, heat-stable substance. It caused dose-dependent inhibition of Na+-independent [3H]GABA bindings. Scatchard plot analysis of the [3H]GABA binding to GABA “B” receptor recognition site showed this inhibitor increased the Kd value (24.1 nM to 3.6 nM) without changing the Bmax. On the other hand, Scatchard plot analysis of the [3H]GABA binding to GABA “A” receptor recognition site showed that the inhibitor decreased number of binding sites (706 fmol/mg protein to 494 fmol/mg protein) without affecting the Kd value. These results suggest that the endogenous inhibitor functions as a modulator for GABAB and GABAA receptors.  相似文献   

20.
Summary We previously reported that3H-folate uptake by rabbit jejunal brush-border membrane (BBM) vesicles was markedly stimulated by an outwardly directed OH gradient (pHin 7.7, pHout 5.5), inhibited by anion exchange inhibitors (DIDS, SITS, furosemide), and saturable (folateK m=0.19 m) suggesting carrier-mediated folate/OH exchange (or H+/folate cotransport). In the present study, the anion specificity of this transport process was examined. Under conditions of an outwardly directed OH gradient, DIDS-sensitive folate uptake wascis inhibited (>90%) by reduced folate analogues: dihydrofolate (IC50=0.40 m), folinic acid (IC50=0.50 m), 5-methyltetrahydrofolate (IC50=0.53 m), and (+)amethopterin (IC50=0.93 M). In contrast, 10 m (–)amethopterin had only a modest effect on folate uptake (18% inhibition) suggesting stereospecificity of the folate/OH exchanger. The nonpteridine compounds which are transported by the folate carrier in L1210 leukemic cells (phthalate, thiamine pyrophosphate, and PO 4 –3 ) did not inhibit jejunal folate uptake. Furthermore, folate uptake was not inhibited by SO 4 –2 (4mm) or oxalate (4mm) thereby distinguishing this carrier from the previously described intestinal SO 4 –2 /OH and oxalate/Cl exchangers. After BBM vesicles were loaded with3H-folate, the initial velocity of3H-folate efflux was stimulated by unlabeled folate in the efflux medium. The transstimulation of3H-folate efflux by unlabeled folate was furosemide (or DIDS) inhibitable and temperature sensitive. Half-maximal stimulation of furosemide-sensitive3H-folate efflux was observed with 0.25±0.05 m unlabeled folate, a concentration similar to theK m for folate uptake. These data suggest that folate-stimulated3H-folate efflux is mediated by the folate/OH exchanger. With the exception of (–) amethopterin, reduced folate analogues also transstimulated furosemide-sensitive3H-folate efflux in a concentration-dependent manner suggesting stereospecific transport of these analogues by the folate/OH exchanger. In summary, folate transport by the jejunal folate/OH exchanger demonstrates bothcis inhibition and transstimulation by reduced folate analogues, but not by other inorganic or organic anions suggesting bidirectional transport of folate and a high degree of anion specificity.  相似文献   

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