Sodium-dependent phosphate transport by apical membrane vesicles from a cultured renal epithelial cell line (LLC-PK1)

Apical membrane vesicles were prepared from confluent monolayers of LLC-PK1 cells grown upon microcarrier beads. The final membrane preparation, obtained by a modified divalent cation precipitation technique, was enriched in alkaline phosphatase, leucine aminopeptidase and trehalase (8-fold compared to the initial homogenate). Analysis of phosphate uptake into the vesicles identified a specific sodium-dependent pathway. Lithium and other cations were unable to replace sodium. At 100 mmol/l sodium and pH 7.4, an apparent Km for phosphate of 99 +/- 19 mumol/l and an apparent Ki for arsenate of 1.9 mmol/l were found. Analysis of the sodium activation of phosphate uptake gave an apparent Km for sodium of 32 +/- 12 mmol/l and suggested the involvement of two sodium ions in the transport mechanism. Sodium modified the apparent Km of the transport system for phosphate. The rate of sodium-dependent phosphate uptake was higher at pH 6.4 than at pH 7.4. At both pH values, an inside negative membrane potential (potassium gradient plus valinomycin) had no stimulatory effect on the rate of the sodium-dependent component of phosphate uptake. It is concluded that the apical membrane of LLC-PK1 cells contains a sodium-phosphate cotransport system with a stoichiometry of 2 sodium ions: 1 phosphate anion.     

Hereditary folate malabsorption: A positively charged amino acid at position 113 of the proton-coupled folate transporter (PCFT/SLC46A1) is required for folic acid binding

The proton-coupled folate transporter (PCFT/SLC46A1) mediates intestinal folate uptake at acidic pH. Some loss of folic acid (FA) transport mutations in PCFT from hereditary folate malabsorption (HFM) patients cluster in R113, thereby suggesting a functional role for this residue. Herein, unlike non-conservative substitutions, an R113H mutant displayed 80-fold increase in the FA transport Km while retaining parental Vmax, hence indicating a major fall in folate substrate affinity. Furthermore, consistent with the preservation of 9% of parental transport activity, R113H transfectants displayed a substantial decrease in the FA growth requirement relative to mock transfectants. Homology modeling based on the crystal structures of the Escherichia coli transporter homologues EmrD and glycerol-3-phosphate transporter revealed that the R113H rotamer properly protrudes into the cytoplasmic face of the minor cleft normally occupied by R113. These findings constitute the first demonstration that a basic amino acid at position 113 is required for folate substrate binding.     

Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.     

An endogenous carrier-mediated uptake system for folate in oocytes of Xenopus laevis.

We investigated the existence of an endogenous uptake system for folate in Xenopus laevis oocytes. This was done by performing uptake measurements using [3H]folic acid. Uptake of folic acid was linear with time for 4 h of incubation, and was similar in collagenase-treated and non-treated oocytes. The uptake process was carrier-mediated, as suggested by the saturation of folic acid uptake with concentration, and by the ability of unlabelled folic acid and its related compounds to significantly inhibit the uptake of [3H]folic acid. The apparent Km and Vmax of the uptake process were 42 +/- 7 nM and 10.56 +/- 0.46 fmol per oocyte per 2 h, respectively. The uptake of folic acid was independent of the presence of Na+ in the incubation medium, but was highly pH dependent with severe inhibition occurring at pH lower than 6.5. Folic acid uptake was energy- and temperature-dependent, and was significantly inhibited by the anion transport inhibitors DIDS and SITS. These results demonstrate the existence of an endogenous carrier-mediated system for folic acid uptake in Xenopus oocytes. Further characterization of the molecular mechanism of folic acid uptake and its regulation in this non mammalian in vitro unicellular system may prove useful in furthering our understanding of folate movement across biological membranes.     

Studies on the thermotropic behavior of aqueous phosphatidylethanolamines

Transport of phosphate has been studied in subconfluent monolayers of LLC-PK1 cells. It was found that this transport system shows similar characteristics to those observed in the kidney. Uptake of phosphate is mediated by a Na+-dependent, substrate-saturable process with an apparent Km value for phosphate of 96 +/- 15 mumol/l. Kinetic analysis of the effect of Na+ indicated that at (pH 7.4) two sodium ions are cotransported with one HOP4(2-) ion (Hill coefficient 1.5) with an apparent Km value for sodium of 56 mmol/l. Pi uptake is inhibited by metabolic inhibitors (ouabain and FCCP). In the pH range of 6.6 of 7.4 Pi uptake rate does not change significantly, indicating that both the monovalent and the divalent form of phosphate are accepted by the transport system. It is suggested that phosphate is transported by LLC-PK1 cells together with sodium (2 Na+:1 HPO4(2-) in an electroneutral manner down a favourable sodium gradient.     

Expression of the reduced folate carrier SLC19A1 in IEC-6 cells results in two distinct transport activities

Intestinal absorption offolates has been characterized as a facilitative process with a low pHoptimum. Studies with intestinal epithelial cells have suggested thatthis activity is mediated by the reduced folate carrier (RFC1). In thispaper, we report on folate transport characteristics in an immortalizedrat IEC-6 cell line that was found to exhibit the predominant influxactivity for methotrexate (MTX) at pH 5.5 with a low level of activity at pH 7.4. Transfection of this cell line with an RFC1 construct resulted in clones exhibiting increased MTX uptake at both the pHs andhigh folic acid uptake only at the low pH. For the two clones with thehighest level of transport activity, relative MTX influx at the two pHswas reversed. Moreover, the low pH MTX influx activity([MTX]_e = 0.5 µM) was markedly inhibited by 20 µM folic acid while influx at neutral pH was not. Furthermore, in thepresence and absence of glucose at low pH, MTX and folic acid influxactivity was inhibited by azide, while MTX influx at pH 7.4 wasstimulated by azide in the absence of glucose but was unchanged in thepresence of glucose and azide. This was contrasted with the results oftransfection of the same RFC1 construct into an L1210 murine leukemiacell line bearing a nonfunctional endogenous carrier. In this case, theactivity expressed was only at pH 7.4. These data indicate that RFC1can exhibit two distinct types of folate transport activities inintestinal cells that must depend on tissue-specific modulators.

     

Na+ and pH dependence of 5-methyltetrahydrofolic acid and methotrexate transport in freshly isolated hepatocytes

The dependence of the high-affinity transport systems for 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) and methotrexate on sodium ions and on pH was examined in freshly isolated rat hepatocytes. Previous studies indicated that transport of these folate derivatives was sodium-dependent. Experiments to determine the Km for sodium of 5-CH3-H4PteGlu transport showed no dependence on extracellular sodium. However, uptake was sodium-dependent when hepatocytes were preincubated for 30 min in sodium-free medium, a treatment which resulted in an increase in the transmembrane pH gradient (delta pH = pH out-pH in) and a decrease in the uptake of 5-CH3-H4PteGlu. Uptake of methotrexate displayed a linear dependence on extracellular sodium ions. Uptake of 5-CH3-H4PteGlu increased linearly as the transmembrane pH gradient decreased; i.e., as the medium became more acid with respect to the cytosol. Lineweaver-Burk and Scatchard plots of 5-CH3-H4PteGlu uptake indicated an apparent Km for H+ of about 24 nM, equivalent to a pH of 7.6. Hill-plots suggested a stoichiometry of 1:1 for the interaction of protons with the 5-CH3-H4PteGlu transport system. Both the Km and Vmax for 5-CH3-H4PteGlu transport were increased at pH 5.5 compared to pH 7.4, suggesting that extracellular protons increased the number of and/or the activity of the membrane carrier. In contrast, methotrexate transport was maximal at pH 7 where the transmembrane pH gradient was zero. These results suggest the possibility that 5-CH3-H4PteGlu may be cotransported along with H+ ions in hepatocytes, although they do not rule out a 'catalytic coupling' whereby protons interact with the carrier to stimulate substrate flux without concomitant H+ transport.     

Folate Transport by Prawn Hepatopancreas Brush-Border Membrane Vesicles

The transport system of folic acid (Pte-Glu) by brush-border membrane vesicles (BBMV) isolated from prawn (Penaeus japonicm) hepatopancreas, was studied by measuring the uptake of Pte-Glu. This uptake was found to have two components, intravesicular transport and membrane binding. Membrane binding was not affected by the presence of a transmembrane pH-gradient at a short incubation period. However, a transmembrane pH-gradient increased membrane binding at 60 min. The transport of Pte-Glu appeared to be carrier-mediated, was stimulated by an inwardly proton gradient (pH 5.5 outside, 7.4 inside) and was unaffected by a sodium-gradient. The relationship between pH gradient-driven Pte-Glu uptake and medium Pte-Glu concentration followed saturating Michaelis–Menten kinetics. Eadie–Hofstee representation of the pH gradient-driven Pte-Glu uptake indicated a single transport system with a Km of 0.37 M and Vmax of 1.06 pmol/mg protein/15 s. These findings indicate that BBMV isolated from prawn hepatopancreas possesses a Pte-Glu transport system similar to that described in mammalian intestine.     

Transport of glucose and fructose in rat hepatocytes at 37 degrees C

The kinetic parameters for transport of the nonmetabolizable glucose analogue 3-O-methyl-D-glucose and the relationship between transport and metabolism of D-glucose and D-fructose were determined in isolated rat hepatocytes at 37 degrees C and pH 7.4. 3-O-Methylglucose at a very low concentration (0.1 mM) equilibrated with the intracellular water with a rate constant of 0.41 s-1. Km for equilibrium exchange entry was 5.5 mM and Vmax was 2.2 mM X s-1 and similar results were obtained when using the zero-trans entry protocol. The rate constant for entry of tracer D-glucose was 0.15 s-1 and Km for glucose was about 20 mM. The phosphorylation rate for D-glucose was much slower than the transport rate. The rate constant for D-fructose entry was about 0.04 s-1, the apparent Km was about 100 mM and Vmax about 5 mM X s-1. The concentration dependence of 3-O-methylglucose inhibition of labelled fructose transport revealed biphasic kinetics indicating that fructose was transferred by both the glucose transporter and a fructose transporter. At concentrations lower than 1 mM, fructose metabolism appeared to be limited by the transport step.     

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model

Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor α (FRα) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro . Two distinct processes are apparent; one that is FRα dependent and one that is independent of the receptor. FRα binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRα independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRα -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remains non-metabolized, supporting the hypothesis that FRα may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRα-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRα.     

Role of reduced folate carrier in intestinal folate uptake

Studies from our laboratory and others have characterized different aspects of the intestinal folate uptake process and have shown that the reduced folate carrier (RFC) is expressed in the gut and plays a role in the uptake process. Little, however, is known about the actual contribution of the RFC system toward total folate uptake by the enterocytes. Addressing this issue in RFC knockout mice is not possible due to the embryonic lethality of the model. In this study, we describe the use of the new approach of lentivirus-mediated short hairpin RNA (shRNA) to selectively silence the endogenous RFC of the rat-derived intestinal epithelial cells (IEC-6), an established in vitro model for folate uptake, and examined the effect of such silencing on folate uptake. First we confirmed that the initial rate of [(3)H]folic acid uptake by IEC-6 cells was pH dependent with a markedly higher uptake at acidic compared with alkaline pH. We also showed that the addition of unlabeled folic acid to the incubation buffer leads to a severe inhibition ( approximately 95%) in [(3)H]folic acid (16 nM) uptake at buffer pH 5.5 but not at buffer pH 7.4. We then examined the effect of treating (for 72 h) IEC-6 cells with RFC-specific shRNA on the levels of RFC protein and mRNA and observed substantial reduction in the levels of both parameters ( approximately 80 and 78%, respectively). Such a treatment was also found to lead to a severe inhibition ( approximately 90%) in initial rate of folate uptake at buffer pH 5.5 (but not at pH 7.4); uptake of the unrelated vitamin, biotin, on the other hand, was not affected by such a treatment. These results demonstrate that the RFC system is the major (if not the only) folate uptake system that is functional in intestinal epithelial cells.     

Chinese hamster ovary cell mutants deficient in an anion exchanger functionally similar to the erythroid band 3

Studies in Chinese hamster ovary cells demonstrate the presence of an anion exchanger, which has some of the properties of the band 3 transporter in erythrocytes. 1) Extracellular chloride is a competitive inhibitor of sulfate influx and stimulates sulfate efflux, suggesting that the mechanism of uptake is SO2-(4)/Cl- exchange. 2) The anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits sulfate uptake in a dose-dependent manner. Half-maximal inhibition is achieved at 0.06 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. 3) Low extracellular pH markedly stimulates sulfate uptake. A 6-fold decrease in the apparent Km is observed at pHout 5.5 as compared to pHout 7.5. However, studies carried out over a broad range of extracellular SO2-(4) concentrations indicate the presence of three components of this transport activity in Chinese hamster ovary cells: two high affinity low capacity systems, one in the range 0.5 microM less than [SO2-(4)]out less than 50 microM and one in the range 50 microM less than [SO2-(4)]out less than 150 microM, and a low affinity, high capacity system (at [SO2-(4)]out greater than 150 microM). These properties have not been previously reported for the erythroid band 3 transporter. The availability of mutants deficient in these activities has enabled us to carry out studies which suggest that the high affinity systems are functionally independent of the low affinity system, but that all systems are dependent on the same anion exchange protein. Studies in a mutant which lacks all components of the transport activity indicates that the anion exchanger may be instrumental in the regulation of the intracellular pH in Chinese hamster ovary cells.     

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

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)     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).     

Comparison of folic acid uptake characteristics by human placental choriocarcinoma cells at acidic and physiological pH

The aim of this work was to characterize the placental uptake of folic acid from the maternal circulation. Using 2 human trophoblast cell lines (BeWo and JAR), we verified that uptake of 3H-folic acid was pH-dependent, increasing significantly with decreasing extracellular pH. In BeWo cells, uptake of 3H-folic acid at pH 5.5 was (i) Na+-independent; (ii) inhibited by folic acid, 5-methyltetrahydrofolate (5-MTHF), and methotrexate (MTX); (iii) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS); (iv) inhibited by the proton ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP); (v) not inhibited by blockers of receptor-mediated endocytosis (cytochalasin D and monensin); (vi) trans-inhibited by MTX and folic acid; and (vii) not affected by an anti-reduced folate transporter-1 (RFC) antibody. At pH 7.5, uptake of 3H-folic acid was (i) Na+-independent; (ii) inhibited by folic acid and MTX, but not by 5-MTHF; (iii) inhibited by SITS, but not by DIDS; (iv) not affected by FCCP; (v) inhibited by monensin (but not by cytochalasin D); (vi) trans-inhibited by folic acid (but not by MTX); and (vii) inhibited by an anti-RFC antibody. In conclusion, in BeWo cells, both RFC and receptor-mediated endocytosis seem to be involved in 3H-folic acid uptake at pH 7.5, whereas at pH 5.5, RFC and (or) a low pH-operating transporter distinct from RFC are involved.     

Ascorbic acid transport and accumulation in human neutrophils

The transport, accumulation, and distribution of ascorbic acid were investigated in isolated human neutrophils utilizing a new ascorbic acid assay, which combined the techniques of high performance liquid chromatography and coulometric electrochemical detection. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized greater than or equal to 94% to the cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Upon addition of ascorbic acid to the extracellular medium in physiologic amounts, ascorbic acid was accumulated in neutrophils in millimolar concentrations. Accumulation was mediated by a high affinity and a low affinity transporter; both transporters were responsible for maintenance of concentration gradients as large as 50-fold. The high affinity transporter had an apparent Km of 2-5 microns by Lineweaver-Burk and Eadie-Hofstee analyses, and the low affinity transporter had an apparent Km of 6-7 mM by similar analyses. Each transporter was saturable and temperature dependent. In normal human blood the high affinity transporter should be saturated, whereas the low affinity transporter should be in its linear phase of uptake.     

Cation and harmaline interactions with Na(+)-independent dibasic amino acid transport system y+ in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish (Eptatretus stouti).

Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.     

Stereoselectivity of the folate transporter in rabbit small intestine: studies with amethopterin enantiomers

Stereoselectivity of the folate transporter was examined using rabbit intestinal brush border membrane vesicles (BBMV). Methotrexate (MTX) and the antipode (D-amethopterin) were used as model substrates of the transporter. Folic acid (FA) and MTX were actively taken up into BBMV in the presence of an H+ gradient. Initial uptake of FA and MTX was concentration-dependent with Km values of 1.5 and 1.6 microM for FA and MTX, respectively. FA and MTX mutually inhibited uptake in a competitive manner, with Ki values being similar to the corresponding Km values, demonstrating that FA and MTX share the folate transporter. D-Amethopterin also inhibited FA uptake competitively, with a Ki value approximately 60-fold greater than that of MTX, showing that the affinity of the D-isomer (D-amethopterin) to the folate transporter is much less than that of the L-isomer (MTX). The extent of stereoselectivity observed in the present study is consistent with the previously reported differences in plasma concentration between amethopterin enantiomers following oral administration in humans.     

L(+)-lactate transport in perfused rat skeletal muscle: kinetic characteristics and sensitivity to pH and transport inhibitors

We have examined lactate uptake (as the rate of net muscle lactate accumulation) and unidirectional inward transport (measured by a paired-tracer dilution method) in muscle of the perfused skinned rat hindlimb. Inhibition of tracer influx (fractional uptake at 1 mM L(+)-lactate, 43.3 +/- 3.1% but only 32.9 +/- 1.8% at 50 mM lactate) suggested some competition between tracer and native forms of the carboxylate for transport. D(-)-lactate (50 mM) did not inhibit uptake of tracer L(+)-lactate. Pyruvate (25 mM), but none of five other monocarboxylates, inhibited uptake of tracer lactate, by 22% (P less than 0.01). Altering perfusate pH from 7.4 to 6.8 caused a 36% increase (P less than 0.001) in the unidirectional L(+)-lactate transport at 1 mM L(+)-lactate, whereas increasing pH to 7.7 reduced transport by 18% (P less than 0.01). Tracer lactate influx was inhibited by 500 microM 4-acetamido-4'-isothiocyanostilbene (SITS) (19%), 5 mM alpha-cyano-4-hydroxycinnamic acid (CIN) (20-30%), 1 mM amiloride (27%) and by a thiol group reagent p-chloromercuribenzenesulphonic acid (pCMBS) (26%). Overall the results indicate that at least two processes are involved in the transfer of lactate: one, saturable, with a Vmax of 0.84 mumol.min-1.g-1 and an apparent Km of 21 mM was sensitive to SITS, CIN, and a thiol group reagent; the other was non-saturable and insensitive to SITS and CIN with an apparent rate constant of 0.1 min-1.     

Kinetic properties of the reconstituted glucose transporter from human erythrocytes

The kinetic parameters of D-glucose transport in liposomes reconstituted with the purified glucose transporter were determined. Net uptake and efflux both had Km values of 0.7 to 1.2 mM and Vmax values of 1.6 mumol/mg of protein/min. Equilibrium exchange had a Km of 35 mM and a Vmax of 50 mumol/mg of protein/min. By separating the liposomes from unreconstituted protein using density centrifugation, the Vmax of exchange was increased to 86 mumol/mg of protein/min, about 3 times that of the erythrocyte membrane. Trypsin, which inhibits erythrocyte glucose transport only from the cytoplasmic side, inhibited reconstituted transport activity about 40% when added externally. With internal treatment as well, the inhibition was about 80%. This suggests that the reconstituted transporter is oriented about equally in both directions. Antibody prepared against the purified transporter inhibits transport to a maximum of about 50%, also indicating a scrambled orientation. External trypsin treatment decreased the Km for uptake and increased the Km for efflux, consistent with asymmetric kinetic parameters for the two faces of the transporter. However, the calculated Km values are lower than those reported for erythrocytes. Phloretin and diethylstilbestrol inhibit the reconstituted transporter. However, they bind to liposomes, producing anomalous results under some experimental conditions. When this binding is taken into account, phloretin inhibits completely and symmetrically. The binding accounts for the apparent asymmetric effects of phloretin reported by others. The inhibitory effects of mercuric ions are consistent with action at two classes of binding sites. Treatment with trypsin increases the sensitivity to Hg2+, indicating that the more sensitive site is on the external face of the transporter.