Intracellular phosphate and its possible role as an exchange anion for active transport of methotrexate in L1210 cells

L1210 cells transport P_i in the absence of added Na⁺. Uptake shows saturation kinetics (K_t = 1.7 mM), is temperature-dependent, and can be reduced 80% by high levels of unlabeled P_i, and thus has the characteristics of a carrier-mediated process. This transport process is also inhibited by methotrexate. The methotrexate-sensitive component constitutes half of total P_i uptake, and is reduced by 50% at a concentration of methotrexate (2 μM) that is comparable to its K_t (1.5 μM) for transport into the cells. An impermeable fluorescent analog of methotrexate and an irreversible inhibitor of the methotrexate transport system (carbodiimide-activated methotrexate) also inhibit this same P_i uptake component. It is concluded that methotrexate and P_i can be transported by the same carrier system. The basis for this shared uptake is suggested to be that the methotrexate carrier protein facilitates the obligatory exchange of extracellular folate compounds for intracellular divalent anions, and that a primary exchange anion is P_i. A principal energy source for active transport of methotrexate might then be the concentration gradient for P_i that is maintained by the Na⁺-dependent, P_i transport system of these cells.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

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.     

Inhibition of intestinal absorption of 5-methyltetrahydrofolate by fluoxetine

Thein vitro uptake of 5-methyltetrahydrofolate (5-MeTHF) by rat and human intestine is dose-dependently inhibited by the antidepressant drug fluoxetine (FLX). In rat jejunum rings, 0.2 mM FLX inhibited the uptake of 5-MeTHF (0.25 μM) by 32% (15 min) and 49% (45 min). In brush border membrane vesicles (BBMV) from rat jejunum, 0.2 mM FLX inhibited the folate uptake at the overshoot (90 s) by 40 %. Similar inhibition was observed with human Caco-2 cells and duodenal biopsies. FLX action is exerted on the active transport component of the folate uptake, since the drug has no effect when the passive diffusion component becomes prominent by high substrate concentration, or by 0-4 ºC incubation or by addition of the folate transport inhibitor DIDS (1mM). The kinetic analysis with rat BBMV suggests a non-competitive inhibition of the 5-MeTHF transport by FLX, with apparent values for K_M = 0.89 μM, Vmax = 1.89 pmol/mg prot./10 s, and K_I = 0.21 mM. After 21 days of treatment with FLX (10 mg/kg/day), the folate uptake by jejunum rings or by BBMV from the treated rats was diminished, and the folate levels in erythrocytes and serum were also decreased.     

Characterization of the multiple transport routes for methotrexate in L1210 cells using phthalate as a model anion substrate

Summary o-Phthalate is actively transported into L1210 cells and the primary route for cell entry is the same transport system which mediates the influx of methotrexate and other folate compounds. The identity of the influx route has been established by the following observations: (A) Phthalate influx is competitively inhibited by methotrexate and the inhibition constant (K _i ) is comparable to theK _i for half-maximal influx of methotrexate; (B) Various anions inhibit the influx of phthalate and methotrexate with comparableK _i values; (C) The influx of phthalate and methotrexate both fluctuate in parallel with changes in the anionic composition of the external medium; and (D) A specific covalent inhibitor of the methotrexate transport system (NHS-methotrexate) also blocks the transport of phthalate. In contrast, the efflux of phthalate does not occur via the methotrexate influx carrier, but rather by two separate processes which can be distinguished by their sensitivities to bromosulfophthalein. Efflux via the bromosulfophthalein-sensitive route constitutes 75% of total efflux and is enhanced by glucose and inhibited by oligomycin. The inability of phthalate to exit via the methotrexate influx carrier is due to competing intracellular anions which prevent phthalate from interacting with the methotrexate binding site at the inner membrane surface.     

Studies of the lungs in diabetes mellitus. II. Phospholipid analyses on the surfactant from broncho-alveolar lavage fluid of alloxan-induced diabetic rats

Structurally diverse anions (folate, 5-formyltetrahydrofolate, AMP, ADP, thiamine pyrophosphate, phosphate, sulfate, and chloride) that are competitive inhibitors of methotrexate influx in L1210 cells also enhance the efflux of methotrexate from these cells. The increase in efflux reaches a maximum of 2- to 4-fold depending upon the anion employed, and the anion concentrations required for half-maximal stimulation of efflux are similar to their K_i values for inhibition of methotrexate influx. A competitive inhibitor of methotrexate uptake (fluorescein-diaminopentane-methotrexate) that is not transported by this system, does not increase methotrexate efflux. These results suggest that the efflux of intracellular methotrexate is coupled to the concomitant uptake of an extracellular anion.     

Transport of methotrexate in L1210 cells: Effect of ions on the rate and extent of uptake

Transport of methotrexate (MTX) in L1210 cells is highly dependent upon the ionic composition of the external medium. Half-maximal rates of MTX transport (K_t values) vary from 0.9 μm in cells suspended in potassium-Hepes buffer containing Mg²⁺ (Hepes-Mg), to 10 μm in phosphate-buffered saline (PBS). At saturating levels of substrate, however, transport rates approach the same maximum velocity (V) regardless of the buffering medium. The increased K_t value for MTX in PBS is due to the presence of the competitive inhibitors, phosphate (K_i = 0.87 mM) and Cl^? (K_i = 46 mM). Concentration gradients for MTX at the steady state are also much lower (about 20-fold) in PBS than in Hepes-Mg; the components of PBS that reduce this uptake parameter are phosphate, Cl^?, Ca²⁺, and Na⁺. Ions that decrease the influx rate or the steady-state level also produce an increase in MTX efflux. Glucose (which increases ATP levels) reduces influx rates and steady-state levels of MTX, and induces efflux in both PBS and Hepes-Mg. Conversely, the combination of azide plus iodoacetate (which reduces ATP levels) stimulates MTX uptake in PBS, but has little effect on MTX transport parameters in Hepes-Mg. The unusually high sensitivity of MTX transport to various anions is consistent with the hypothesis that this system catalyzes the exchange of external MTX for an intracellular anion, and that efflux of the anion down a concentration gradient provides the driving force for active transport of MTX.     

Glucose transport and metabolism in Gymnodinium breve

Florida's red tide organism, Gymnodinium breve, utilized exogenous glucose in the light for the synthesis of cellular components. Glucose was not taken up in the dark. Kinetic parameters for glucose uptake include a K_FD of 11 μM and a V_max of 1 × 10^?10 mol of glucose taken up/mg cellular protein/hr. Glucose uptake was competitively inhibited by phloridzin (K_i = 40 μM), mannose (K_i = 12O μM), and 2-deoxy-d-glucose (K_i = 190 μM) and non-competitively inhibited by galactose (K_i = 125 μM). Kinetics and inhibition of glucose uptake are consistent with a facilitated diffusion transport system.     

Structural requirements for anion substrates of the methotrexate transport system in L1210 cells

A broad spectrum of structurally diverse anions reversibly inhibits the influx of methotrexate in L1210 cells. Several of the more effective anions and their respective inhibition constants (K_i values) were: 5-methyltetrahydrofolate (0.3 μm), bromosulfophthalein (2 μm), thiamine pyrophosphate (3 μm), 8-anilino-1-naphthalene sulfonate (7 μm), phthalate (20 μm), and AMP (50 μm). Moderate inhibition was observed with P_i (K_i = 400 μm) and other divalent inorganic anions, while small monovalent anions such as Cl^? (K_i = 30 mm) were the least effective. When these same anions were tested for an effect on methotrexate efflux, stimulation was observed with some anions, while others had no effect. Enhancement was produced by folate compounds and p-aminobenzoylglutamate, small monovalent (e.g., Cl^?, acetate, and lactate) and divalent (e.g., phosphate and succinate) anions, a few nucleotides (e.g., AMP), and thiamine pyrophosphate, while little or no effect was associated with trivalent anions (e.g., citrate), most nucleotides, and large organic anions (e.g., bromosulfophthalein, NAD, and NADP). Anions with the ability to promote methotrexate efflux in control cells lost this capacity upon exposure of the cells to an irreversible inhibitor of methotrexate influx. These results support the hypothesis that methotrexate transport proceeds via an anion-exchange mechanism and moreover provide evidence that anion substrates for this system can be identified by their ability to promote methotrexate efflux. Anions which appear most likely to participate in this exchange cycle in vivo are P_i and AMP.     

Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells

A binding component with a high affinity for 5-methyltetrahydrofolate (K_D = 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 K_D values for 5-methyltetrahydrofolate and methotrexate binding and the K_t 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.     

Transport of methotrexate in L1210 cells: Mechanism for inhibition by p-chloromercuriphenylsulfonate and N-ethylmaleimide

Methotrexate transport in L1210 cells is highly sensitive to inhibition by p-chloromercuriphenylsulfonate (CMPS) and, to a lesser extent, by N-ethylmaleimide. A 50% reduction in the methotrexate influx rate occurred upon exposure of cells to 3 μM CMPS or 175 μM N-ethylmaleimide, while complete inhibition was achieved at higher levels of these agents. Dithiothreitol reversed the inhibition by CMPS, suggesting that a sulfhydryl residue is involved. This residue is apparently not located at the substrate binding site of the transport protein, since methotrexate failed to protect the system from inactivation by either CMPS or N-ethylmaleimide, and the transport protein retained the ability to bind substrate (at 4°C) after exposure to these inhibitors (at 37°C). Methotrexate efflux was also inhibited by CMPS (50% at 4 μM), indicating that both the uptake and efflux of methotrexate in L1210 cells occur via the same transport system. High concentrations of CMPS (greater than 20 μM) increased the efflux rate, apparently by damaging the cell membrane and allowing the passive diffusion of methotrexate out of the cell.     

Adenine nucleotide metabolism of blood platelets. VIII. Transport of adenine into human platelets

Adenine uptake into human blood platelets is a carrier-mediated process with a K_m of 159±21 nM and a V of 100±10 pmoles/min per 10⁹ platelets (in citrated platelet-rich plasma). The Q₁₀ was 2.53±0.22. A pH optimum was found at 7.5. Washing of the platelets increased the K_m to 453±33 nM and V to 397±38 pmoles/min per 10⁹ platelets. The change in shape induced in platelets by ADP was accompanied by an increase in V (2 times) and K_m (1.5 times).Guanine (K_i 50 μM), hypoxanthine (K_i 390 μM), adenine-N′-oxide (K_i 40 μM), adenosine (K_i 100 μM), RA 233 (K_i 75 μM) and papaverine (K_i 15 μM) acted as competitive inhibitors. Adenosine at low concentrations, and prostaglandin E₁ gave inhibition at only high adenine levels. A similar inhibition was obtained with 2-deoxy-d-glucose. Sulfhydryl-group inhibitors, pyrimidines and ouabain had no effect.     

Hypoxanthine transport by Chinese hamster lung fibroblasts: Kinetics and inhibition of nucleosides

The transport of [${}^3\text{H}$]hypoxanthine was studied in monolayer cultures of mutant Chinese hamster lung fibroblasts lacking hypoxanthine-guanine phosphoribosyltransferase. Initial rates of transport were determined by rapid uptake experiments (8 to 20 s); a Michaelis constant of 0.68 ± 0.09 mm for hypoxanthine was derived from linear, monophasic plots of $\text{v}\text{S}$ against v. Nucleosides are competitive inhibitors of this process; adenosine and thymidine give respective K_i values of 86 and 300 μm. The corresponding bases give much higher inhibition constants with adenine and thymine yielding values of 3100 and 1700 μm, respectively. A similar pattern was observed for competitive inhibition of hypoxanthine transport by inosine, adenine arabinoside, uridine, cytidine, and two ribofuranosylimidazo derivatives of pyrimidin-4-one; in every case the nucleoside exhibited a lower K_i value than the corresponding homologous base. The inhibition constants observed for nucleosides are remarkably similar to their K_m values for nucleoside transport by cultured cells recently reported by others. Hypoxanthine transport was also blocked by the 6-(2-hydroxy-5-nitrobenzylthio) derivatives of inosine and guanosine and by dipyridamole; these agents are also inhibitors of nucleoside transport. These results indicate a closer relationship between base and nucleoside transport than previously recognized and suggest that these two transport processes may involve identical or very similar transport proteins.     

Uridine transport in Novikoff rat hepatoma cells and other cell lines and its relationship to uridine phosphorylation and phosphorolysis.

Time courses of [³H]uridine uptake as a function of uridine concentration were determined at 25° in untreated and ATP-depleted wild-type and uridine kinase-deficient Novikoff cells and in mouse L and P388 cells, Chinese hamster ovary cells and human HeLa cells. Short term uptake was measured by a rapid sampling technique which allows sampling of cell suspensions in intervals as short as one and one-half seconds. The initial segments of the time courses were the same in untreated, wild-type cells in which uridine is rapidly phosphorylated and in cells in which uridine phosphorylation was prevented due to lack of ATP or uridine kinase. The initial rates of uptake, therefore, reflected the rate of uridine transport. Uridine uptake, however, was approximately linear for only five to ten seconds at uridine concentrations from 20–160 μM and somewhat longer at higher concentrations. In phosphorylating cells the rate of uridine uptake (at 80 μM) then decreased to about 20–30% of the initial rate and this rate was largely determined by the rate of phosphorylation rather than transport. At uridine concentrations below 1 μM, however, the rate of intracellular phosphorylation in Novikoff cells approached the transport rate. The apparent substrate saturation of phosphorylation suggests the presence of a low K_m uridine phosphorylation system in these cells. The “zero-trans” (zt) K_m for the facilitated transport of uridine as estimated from initial uptake rates fell between 50 and 240 μM for all cell lines examined. The zero-trans V_max values were also similar for all the lines (4–15 pmoles/μ1 cell H₂O.sec). The time courses of uridine uptake by CHO cells and the kinetic constants for transport were about the same whether the cells were propagated (and analyzed for uridine uptake) in suspension or monolayer culture. When Novikoff cells were preloaded with 10 μM uridine the apparent K_m and V_max values (infinite-trans) were two to three times higher than the corresponding zero-trans values. Uridine transport was inhibited in a simple competitive manner by several other ribo- and deoxyribonucleosides. All nucleosides seem to be transported by the same system, but with different efficiencies. Uridine transport was also inhibited by hypoxanthine, adenine, thymine, Persantin, papaverin, and o-nitrobenzylthioinosine, and by pretreatment of the cells with p-chloromercuri-benzoate, but not by high concentrations of cytosine, D-ribose or acronycin. The inhibition of uridine transport by Persantin involved changes in both V and K. Because of the rapidity of transport, some loss of intracellular uridine occurred when cells were rinsed in buffer solution to remove extracellular substrate, even at 0°. This loss was prevented by the presence of a transport inhibitor, Persantin, in the rinse fluid or by separating suspended cells from the medium by centrifugation through oil. Metabolic conversion of intracellular uridine were also found to continue during the rinse period. The extent of artifacts due to efflux and metabolism during rinsing increased with duration of the rinse.     

The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1

This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx K_t at pH 5.5 for H247A was ↓8.4-fold. Although wild type (WT)-PCFT K_i values varied among the folates, K_i values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His²⁴⁷ to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser¹⁷². The folic acid influx K_t for S172A-PCFT was decreased similar to H247A. His²⁸¹ faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ∼12-fold↑ in the folic acid influx K_t. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx K_t was unchanged, but the K_t decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His²⁴⁷ to Ser¹⁷² interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His²⁸¹ residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.     

Nucleoside transport in cultured mammalian cells multiple forms with different sensitivity to inhibition by nitrobenzylthioinosine or hypoxanthine

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 (K_d = 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.     

High-affinity transport and phosphorylation of 2-deoxy-D-glucose in synaptosomes

2-Deoxy-d -glucose (2 DG) entered synaptosomes (from rat brain) by a high-affinity, Na⁺-independent glucose transport system with a K_m, of 0.24 mM. 3-O-methyl-glucose, D-glucose, and phloretin were competitive inhibitors of 2-DG transport with K_i's of 7 mM, 64 μM, and 0·75 μM, respectively. Insulin was without effect. 2-DG uptake was also saturable at high substrate concentrations with an apparent low affinity K_m, of 75 mM, where the K_l, for glucose was 17.5 mM. We are not certain whether the rate-limiting step for the low-affinity uptake system is attributable to transport or phosphorylation. However, the high-affinity glucose transport system probably is a special property of neuronal cell membranes and could be useful in helping to distinguish separated neurons from glial cells.     

Photoinactivation of the methotrexate transport system of L1210 cells by 8-azidoadenosin 5'-monophosphate.

Methotrexate transport in L1210 cells is mediated by a carrier protein that can bind organic and inorganic phosphate compounds in addition to the various folate substrates. The photoaffinity labeling agent, 8-azidoadenosine 5'-monophosphate (8-azido-AMP), also interactis (Ki = 140 microM) with the receptor site for this transport system, and upon irradiation with ultraviolet light, irreversibly inhibits methotrexate uptake. Protection against this inactivation is afforded by either a substrate (methotrexate) or a competitive inhibitor (inorganic phosphate). The light-induced reaction proceeds rapidly (t1/2 = 2 min at 23 degrees C under the conditions described) and produces half-maximal reduction in the transport rate when the 8-azido-AMP concentration is 65 microM. complete photoinactivation of methotrexate transport could not be obtained from a single exposure to 8-azido-AMP (up to 1.0 mM), but it could be achieved by the repetitive illumination of cells in a fresh medium. The phosphate and folate/adenine transport systems of L1210 cells are not affected by irradiation in the presence of 8-azido-AMP.     

Influx and net flux of amino acids into larval and juvenile European flat oysters, Ostrea edulis (L.)

Influx and net flux of amino acids into veliger, pediveliger, and juvenile stages of the oyster, Ostrea edulus (L.), were examined. Influx of alanine and glycine was determined using ¹⁴C-labelled substrates and monitoring the disappearance of radioactivity in the medium. Net influx was determined fluorometrically by following the disappearance of primary amines from solutions containing known substrates. Rates of influx and net influx are comparable for juvenile Ostrea down to ambient substrate levels of 2 μM. Net influx of amino acids into veliger and pediveliger larvae occurs at all concentrations examined (9 μM minimum). Rates of maximum influx (J_maxⁱ) and ambient substrate concentration at which influx is half-maximal (K_t) are reported for each stage. The K_t for glycine and alamine influx is 35–40 μM, decreasing to ≈ 15 μM in 1.5 mm sized juvenile oysters.     

CARRIER MEDIATED GLUCOSE TRANSPORT IN CAPILLARIES ISOLATED FROM RAT BRAIN1

Abstract— Uptake of 2-deoxy-d-glucose (2-DG) was investigated in capillaries isolated from rat brain. A high affinity, carrier-mediated transport system was defined with an apparent Km for 2-DG of 93 μM. Uptake was temperature-dependent and markedly inhibited by phloretin and selected hexose isomers. The apparent K_i for d-glucose inhibition of 2-DG uptake was 98 μM. Essentially all of the 2-DG retained by the capillary preparation could be released by sonication and 95% was recovered as free unphosphorylated 2-DG. Uptake was not sodium-dependent and not altered by insulin. These results suggest that movement of glucose across the blood-brain barrier through endothelial cells probably is not rate-limiting unless blood glucose levels are extremely low.     

Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate

Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (K_i = 15 nM) interacted reversibly with the binding site for folate (K_d = 0.8 nM). Cells treated with activated [³H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.