Involvement of a soybean ATP-binding cassette-type transporter in the secretion of genistein, a signal flavonoid in legume-Rhizobium symbiosis

Legume plants have an ability to fix atmospheric nitrogen into nutrients via symbiosis with soil microbes. As the initial event of the symbiosis, legume plants secrete flavonoids into the rhizosphere to attract rhizobia. Secretion of flavonoids is indispensable for the establishment of symbiotic nitrogen fixation, but almost nothing is known about the membrane transport mechanism of flavonoid secretion from legume root cells. In this study, we performed biochemical analyses to characterize the transport mechanism of flavonoid secretion using soybean (Glycine max) in which genistein is a signal flavonoid. Plasma membrane vesicles prepared from soybean roots showed clear transport activity of genistein in an ATP-dependent manner. This transport activity was inhibited by sodium orthovanadate, a typical inhibitor of ATP-binding cassette (ABC) transporters, but was hardly affected by various ionophores, such as gramicidin D, nigericin, or valinomycin, suggesting involvement of an ABC transporter in the secretion of flavonoids from soybean roots. The K(m) and V(max) values of this transport were calculated to be 158 mum and 322 pmol mg protein(-1) min(-1), respectively. Competition experiments using various flavonoids of both aglycone and glucoside varieties suggested that this ABC-type transporter recognizes genistein and daidzein, another signaling compound in soybean root exudates, as well as other isoflavonoid aglycones as its substrates. Transport activity was constitutive regardless of the availability of nitrogen nutrition. This is, to our knowledge, the first biochemical characterization of the membrane transport of flavonoid secretion from roots.     

Lateral diffusion as a rate-limiting step in ubiquinone-mediated mitochondrial electron transport

Data are presented which indicate that the diffusion-based collisions of ubiquinone with its redox partners in the mitochondrial inner membrane are a rate-limiting step for maximum (uncoupled) rates of succinate-linked electron transport. Data were obtained from experimental analysis of a comparison of the apparent activation energies of lateral diffusion rates, collision frequencies, and electron transport rates in native and protein-diluted (phospholipid-enriched) inner membranes. Diffusion coefficients for Complex III (ubiquinol:cytochrome c oxidoreductase) and ubiquinone redox components were determined as a function of temperature using fluorescence recovery after photobleaching, and collision frequencies of appropriate redox partners were subsequently calculated. The data reveal that 1) the apparent activation energies for both diffusion and electron transport were highest in the native inner membrane and decreased with decreasing protein density, 2) the apparent activation energy for the diffusion step of ubiquinone made up the most significant portion of the activation energy for the overall kinetic activity, i.e. electron transport steps plus the diffusion steps, 3) the apparent activation energies for both diffusion and electron transport decreased in a proportionate manner as the membrane protein density was decreased, and 4) Arrhenius plots of the ratio of experimental electron transport productive collisions (turnovers) to calculated theoretically predicted, diffusion-based collisions for ubiquinone with its redox partners had little or no temperature dependence, indicating that as temperature increases, increases in electron transport rate are accounted for by the increases in diffusion-based collisions. These data support the Random Collision Model of mitochondrial electron transport in which the rates of diffusion and appropriate concentrations of redox components limit the maximum rates of electron transport in the inner membrane.     

Dietary polyphenols decrease glucose uptake by human intestinal Caco-2 cells

The effect of different classes of dietary polyphenols on intestinal glucose uptake was investigated using polarised Caco-2 intestinal cells. Glucose uptake into cells under sodium-dependent conditions was inhibited by flavonoid glycosides and non-glycosylated polyphenols whereas aglycones and phenolic acids were without effect. Under sodium-free conditions, aglycones and non-glycosylated polyphenols inhibited glucose uptake whereas glycosides and phenolic acids were ineffective. These data suggest that aglycones inhibit facilitated glucose uptake whereas glycosides inhibit the active transport of glucose. The non-glycosylated dietary polyphenols appear to exert their effects via steric hindrance, and (-)-epigallochatechingallate, (-)-epichatechingallate and (-)-epigallochatechin are effective against both transporters.     

Intestinal permeability of antivirus constituents from the fruits of Eucalyptus globulus Labill. in Caco-2 Cell Model

The uptake and transepithelial transport of the three main constituents macrocarpal A (M-A), macrocarpal B (M-B), and cypellocarpa C (Cy-C) from the fruits of Eucalyptus globulus Labill. were investigated. Monolayers of the human intestinal epithelial cancer cell line Caco-2 were incubated with M-A, M-B, and Cy-C to model its intestinal absorption and transport, respectively. The determination of compounds was performed by HPLC. The apparent permeability coefficients (P(app)) for M-A, M-B, and Cy-C in the apical-to-basolateral direction of a Caco-2 monolayer were (1.70+/-0.06)x10(-6), (1.99+/-0.10)x10(-6), and (6.08+/-0.41)x10(-6)cm/s, respectively. In the presence of iodoacetamide, the P(app) of Cy-C were both reducted in apical-to-basolateral and basolateral-to-apical directions. M-A and M-B appear to accumulate in the epithelial cells. The intestinal absorption of M-A, M-B, and Cy-C was passive diffusion as the dominating process and Cy-C was partly ATP-dependent.     

Estimation of apparent L-amino acid diffusion in porcine jejunal enterocyte brush border membrane vesicles

There is an overlap of carrier-mediated L-amino acid transport and apparent simple diffusion when measured in intestinal brush border membrane vesicles. Using L-threonine and L-glutamine as representative amino acids, this study was undertaken to estimate apparent simple diffusion of L-amino acids and to establish the effective dosage of HgCl2 for completely blocking carrier-mediated L-amino acid transport in porcine jejunal enterocyte brush border membrane vesicles. Jejunal mucosa was scraped from three pigs weighing 26 kg. Enterocyte brush border membrane vesicles, with an average enrichment of 24-fold in sucrase specific activity, were prepared by Mg2+-precipitation and differential centrifugation. In vitro uptake was measured by the fast filtration manual procedure. HgCl2 blocked the carrier-mediated initial transport of L-threonine and L-glutamine under Na+-gradient condition in a dose-dependent manner. At the minimal concentration of 0.165 micromol HgCl2 mg(-1) protein, carrier-mediated L-threonine and L-glutamine transport was completely inhibited. The apparent L-threonine and L-glutamine diffusion was estimated to be 8.6+/-0.7 and 12.4+/-1.0% of the total uptake at the substrate concentrations of 5 microM (L-threonine) and 50 microM (L-glutamine). Therefore, the treatment of porcine brush border membrane vesicles with a minimum of 0.165 micromol HgCl2 mg(-1) protein completely blocks carrier-mediated L-amino acid transport and enables the direct estimation of apparent L-amino acid diffusion in enterocyte brush border membrane vesicles.     

Bicarbonate/chloride antiport in Vero cells: II. Mechanisms for bicarbonate-dependent regulation of intracellular pH

The rates of bicarbonate-dependent uptake and efflux of 22Na+ in Vero cells were studied and compared with the uptake and efflux of 36Cl-. Both processes were strongly inhibited by DIDS. Whereas the transport of chloride increased approximately ten-fold when the internal pH was increased over a narrow range around neutrality, the uptake of Na+ was much less affected by changes in pH. The bicarbonate-linked uptake of 22Na+ was dependent on internal Cl- but not on internal Na+. At a constant external concentration of HCO3-, the amount of 22Na+ associated with the cells increased when the internal concentration of HCO3- decreased and vice versa, which is compatible with the possibility that the ion pair NaCO3- is the transported species and that the transport is symmetric across the membrane. Bicarbonate inhibited the uptake of 36Cl- both in the absence and presence of Na+. At alkaline internal pH, HCO3- stimulated the efflux of 36Cl- from preloaded cells, while at acidic internal pH both Na+ and HCO3- were required to induce 36Cl- efflux. We propose a model for how bicarbonate-dependent regulation of the internal pH may occur. This model implies the existence of two bicarbonate transport mechanisms that, under physiological conditions, transport OH(-)-equivalents in opposite directions across the plasma membrane.     

Sodium-coupled sugar and amino acid transport in an acidic microenvironment

1. Nutrient transport mechanisms of lobster hepatopancreatic epithelial brush border membrane vesicles (BBMV) are strongly influenced by the acidic nature of the tubular lumen. 2. Sodium-dependent glucose uptake by BBMV was electrogenic and was stimulated at low pH by reducing sugar transport Ki, without affecting JM. 3. Glutamate was largely transported in zwitterionic form at pH 4.0 by an electrically silent cotransport mechanism with both Na and Cl. 4. Increased H+ concentration tripled the apparent membrane permeability to glutamate as well as the amino acid transport JM. 5. At pH 4.0 leucine was transported as a cation by two dissimilar carrier systems: a Na-independent process shared by polar amino acids, and an electroneutral Na-2Cl-dependent mechanism shared with non-polar amino acids. 6. A model is proposed for hepatopancreatic BBMV at acidic pH which employs ionic chemical gradients and membrane potential as nutrient transport driving forces.     

Temperature dependence of the apparent affinity and the maximum velocity of the membrane-bound monosaccharide transport system in the yeast Rhodotorula gracilis

Analysis of the temperature dependence of the monosaccharide transport system in the yeast Rhodotorula gracilis (ATCC 26194, CBS 6681), as tested with D-xylose, revealed that the apparent affinity of the transport system, measured as the reciprocal of the half-saturation constant KT, increased when transport velocity was stimulated by temperature (15--30 degrees C) and decreased when the rate of uptake was reduced at temperatures aboce 30 degrees C. Breaks in Arrhenius plots were accompanied by corresponding breaks in van't Hoff plots. Whereas untreated cells exhibited in the van't Hoff plot a discontinuity at 28--30 degrees C this was not observed in heat-treated cells (at either 37 or 45 degrees C). In heat-treated cells the maximum transport velocity was always lower and the apparent affinity higher than in untreated cells at the same temperature; the optimum temperature for both transport velocity and apparent affinity was shifted to higher values. The data are interpreted in terms of a reversible phase transition of membrane lipids effecting an irreversible alteration of membrane structure. The temperature-induced reversible alkalinization of unbuffered yeast suspensions supports this interpretation.     

Identification of different transport systems for bile salts in sinusoidal and canalicular membranes of hepatocytes

The preservation of the functional polarity of hepatocytes in liver snips (1 x 2 x 4 mm) was demonstrated by fluorescent microscopic studies using the sodium salt of (N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-3 beta-amino-7 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid. This fluorescent bile salt derivative is not only taken up by hepatocytes of several cell layers at the surface of the snips but also secreted into bile canaliculi. The intact hepatobiliary transport of bile salts by hepatocytes of liver snips demonstrates that they are a useful system for the investigation of those transcellular transport processes which require the integrity of hepatic structure. Photoaffinity labelling of liver snips with the sodium salt of (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan- 24-oyl)-2-aminoethanesulfonic acid revealed that the bile-salt-binding membrane polypeptides with apparent Mr values of 54,000 and 48,000 are exclusively located in the sinusoidal membrane, whereas a single bile-salt-binding polypeptide with an apparent Mr of 100,000 is located in the bile-canalicular membrane. Photoaffinity labelling of liver snips at 4 degrees C, when transcellular bile-salt transport is insignificant, resulted in the labelling of the two sinusoidal membrane polypeptides and practically no labelling of the polypeptide with an apparent Mr of 100,000. This latter polypeptide was also not labelled when Ca2 deprivation abolished bile secretion completely. These results indicate that the directed hepatobiliary transport of bile salts in hepatocytes is accomplished by transport systems which are different for sinusoidal uptake and canalicular secretion.     

The novel correlation of carbonic anhydrase II and anion exchanger 1 in gills of the spotted green pufferfish, Tetraodon nigrovirids

A novel relationship between branchial carbonic anhydrase II (CAII) and anion exchanger 1 (AE1) was investigated in the euryhaline spotted green pufferfish (Tetraodon nigroviridis). The immunoblots revealed that AE1 was only detected in the membrane fraction of gills while CAII can be probed both in the membrane and cytosol fractions of gills. CAII protein abundance in the membrane fraction is salinity dependent. Immunological detection of the membrane fraction CAII protein in gills showed 3.9-fold higher in the hyposmotic (freshwater) group than the hyperosmotic (seawater;35 per thousand) group. In contrast, there was no change in the protein level of cytosolic CAII between seawater and freshwater groups. The whole-mount immunocytochemical staining demonstrated that both AE1 and CAII were colocalized to the Na(+)/K(+)-ATPase-immunoreactive cells in gill epithelium of the pufferfish. The interaction between CAII and AE1 was further identified by co-immunoprecipitation because AE1 was detected in the immunoprecipitates of CAII and vice versa. Our results showed that in pufferfish gills CAII was not only expressed in the cytosol to produce the substrate for AE1 transport during Cl(-) influx but also associated with the plasma membrane via AE1. Obviously, it is essential for the physiological function of AE1 to interact with CAII in the membrane of gill Na(+)/K(+)-ATPase-immunoreactive cells. To our knowledge, this is the first study to demonstrate the interaction of branchial CAII and AE1 in fish. The novel correlation proposed a new model of Cl(-)/HCO(3) (-) transport in gills of the teleosts.     

Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes

The recently identified human and rodent plasma membrane proteins CNT1, CNT2 and CNT3 belong to a gene family (CNT) that also includes the bacterial nucleoside transport protein NupC. Heterologous expression in Xenopus oocytes has established that CNT1-3 correspond functionally to the three major concentrative nucleoside transport processes found in human and other mammalian cells (systems cit, cif and cib, respectively) and mediate Na(+) - linked uptake of both physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs. Here, one describes a complementary Xenopus oocyte transport study of Escherichia coli NupC using the plasmid vector pGEM-HE in which the coding region of NupC was flanked by 5'- and 3'-untranslated sequences from a Xenopus beta-globin gene. Recombinant NupC resembled human (h) and rat (r) CNT1 in nucleoside selectivity, including an ability to transport adenosine and the chemotherapeutic drugs 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxycytidine (ddC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine), but also interacted with inosine and 2',3'- dideoxyinosine (ddl). Apparent affinities were higher than for hCNT1, with apparent K(m) values of 1.5-6.3 microM for adenosine, uridine and gemcitabine, and 112 and 130 microM, respectively, for AZT and ddC. Unlike the relatively low translocation capacity of hCNT1 and rCNT1 for adenosine, NupC exhibited broadly similar apparent V(max) values for adenosine, uridine and nucleoside drugs. NupC did not require Na(+) for activity and was H(+) - dependent. The kinetics of uridine transport measured as a function of external pH were consistent with an ordered transport model in which H(+) binds to the transporter first followed by the nucleoside. These experiments establish the NupC-pGEM-HE/oocyte system as a useful tool for characterization of NupC-mediated transport of physiological nucleosides and clinically relevant nucleoside therapeutic drugs.     

Influx and efflux kinetics of cationic dye binding to respiring mitochondria.

We have investigated the kinetics of interaction of cationic fluorescent lipophiles (dyes) rhodamine 123, rhodamine 6G, tetramethyl rhodamine ethyl ester, safranine O, 1,1'-diethyloxacarbocyanine, 1,1'-diethyloxadicarbocyanine, and 1,1'-diethylthiadicarbocyanine iodide with isolated respiring rat-liver mitochondria (RLM). Dye flux across the RLM inner membrane was measured by following the kinetics of fluorescence signal change after mixing of dye and RLM. The time course of fluorescence was analysed in terms of a kinetic model of the binding and transport processes involved. The rate constants of dye influx and efflux were extracted from the observed effect on the apparent time constant of fluorescence change to equilibrium intensity upon mixing dye with increasing concentrations of RLM. From the influx rate constants obtained, the apparent permeability constants for dye influx (at zero potential) across the membrane were calculated and ranged from 3 to 140 x 10(-4) cm/s. The influx rate constant was found to be linearly related to relative dye lipophilicity, as predicted by the model. As another test of the model, from the ratio of the influx and efflux rate constants, the apparent trans-membrane potential, psi, was calculated and found generally to agree with reported values, but to depend on the lipophilicity of the dye used. Not predicted by the simple model was a dissymmtry observed in the influx and efflux time constants for fluorescence change to equilibrium intensity. Inferences are made relating to the utility of these dyes as probes of psi.     

Characterization of electrogenic bromosulfophthalein transport in carnation petal microsomes and its inhibition by antibodies against bilitranslocase

Bilitranslocase is a rat liver plasma membrane carrier, displaying a high-affinity binding site for bilirubin. It is competitively inhibited by grape anthocyanins, including aglycones and their mono- and di-glycosylated derivatives. In plant cells, anthocyanins are synthesized in the cytoplasm and then translocated into the central vacuole, by mechanisms yet to be fully characterized. The aim of this work was to determine whether a homologue of rat liver bilitranslocase is expressed in carnation petals, where it might play a role in the membrane transport of anthocyanins. The bromosulfophthalein-based assay of rat liver bilitranslocase transport activity was implemented in subcellular membrane fractions, leading to the identification of a bromosulfophthalein carrier (K(M) = 5.3 microm), which is competitively inhibited by cyanidine 3-glucoside (Ki = 51.6 microm) and mainly noncompetitively by cyanidin (Ki = 88.3 microm). Two antisequence antibodies against bilitranslocase inhibited this carrier. In analogy to liver bilitranslocase, one antibody identified a bilirubin-binding site (Kd = 1.7 nm) in the carnation carrier. The other antibody identified a high-affinity binding site for cyanidine 3-glucoside (Kd = 1.7 microm) on the carnation carrier only, and a high-affinity bilirubin-binding site (Kd = 0.33 nm) on the liver carrier only. Immunoblots showed a putative homologue of rat liver bilitranslocase in both plasma membrane and tonoplast fractions, isolated from carnation petals. Furthermore, only epidermal cells were immunolabeled in petal sections examined by microscopy. In conclusion, carnation petals express a homologue of rat liver bilitranslocase, with a putative function in the membrane transport of secondary metabolites.     

Transport of phosphoenolpyruvate through the erythrocyte membrane.

Phosphoenolpyruvate was transported through the erythrocyte membrane at low pH (4.5-6.5). The influx was observed not only in an iso-osmotic sucrose medium, but also in 0.1 M-citrate solution, but it was negligible in an iso-osmotic NaC1 solution. Efflux, however, was observed in both the sucrose and NaC1 solutions. Compounds derived from phosphoenolpyruvate by replacing the methene group by similarly hydrophobic groups such as hydrogen or the methyl group were permeant but those with the hydrophilic hydroxymethyl group were impermeant. This transport was inhibited by the treatment with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid or pyridoxal phosphate/NaBH4, which are known to be specific for the transport of anions such as C1-, SO42- and HPO42-. It showed saturation kinetics with respect to phosphoenolpyruvate concentration in the medium. These results suggest that the transport of phosphoenolpyruvate is mediated by the anion-transport system. Although phosphoenolpyruvate was transported against the concentration gradient, the transport was characterized as a passive transport, and this apparent uphill transport was interpreted by the Donnan equilibrium.     

Facile synthesis of triterpenoid saponins bearing β-Glu/Gal-(1→3)-β-GluA methyl ester and their cytotoxic activities

Convenient synthetic strategy toward spinasaponin A methyl ester 1 and calenduloside G methyl ester 2, two natural oleanane-type triterpenoid saponins bearing an unique β-D-glucosyl/galactosyl-(1→3)-β-D-glucuronic acid methyl ester disaccharide moiety, was established. Based on this facile approach, four structurally modified congeners 3-6 with ursolic acid and glycyrrhetinic acid as aglycones were efficiently synthesized. MTT assay revealed the cytotoxicities against cancer cells of the synthesized saponins were varied with the change of aglycones and sugar units. Saponin 2 possessing the most potent cytotoxic effects could induce apoptosis of MCF-7 cells, which was detected by confocal micrographs using DAPI staining and flow cytometry using Annexin V and PI double staining. Furthermore, 2-induced apoptosis in MCF-7 cells was associated with ROS generation and loss of the mitochondria membrane potential (Δψ(m)).     

The interaction of anthocyanins with bilitranslocase

Bilitranslocase (TC 2.A.65.1.1) is an organic anion membrane carrier expressed at the sinusoidal domain of the liver plasma membrane and in epithelial cells of the gastric mucosa. Its substrates are sulfobromophthalein, bilirubin, and nicotinic acid. This work reports on the identification of a new class of bilitranslocase substrates, i.e., anthocyanins. Seventeen out thes 20 compounds tested behaved as competitive inhibitors of bilitranslocase transport activity (K(I)=1.4-22 microM). Their structure-activity relationship reveals that mono- and di-glucosyl anthocyanins, the anthocyanin species occurring in food, are better ligands than the corresponding aglycones. Moreover, the first interaction of anthocyanins with the carrier occurs through hydrophilic moieties, such as the 3-glucosyl moiety and the B ring for the monoglucosides, through the 5-glucosyl moiety and the A ring for the diglucosides, and through either the B or the A ring for the aglycones. These findings suggest that bilitranslocase could play a role in the bioavailability of anthocyanins.     

Identification and localization of sodium-phosphate cotransporters in hepatocytes and cholangiocytes of rat liver

Hepatocytes and cholangiocytes release ATP into bile, where it is rapidly degraded into adenosine and P(i). In rat, biliary P(i) concentration (0.01 mM) is approximately 100-fold and 200-fold lower than in hepatocytes and plasma, respectively, indicating active reabsorption of biliary P(i). We aimed to functionally characterize canalicular P(i) reabsorption in rat liver and to identify the involved P(i) transport system(s). P(i) transport was determined in isolated rat canalicular liver plasma membrane (LPM) vesicles using a rapid membrane filtration technique. Identification of putative P(i) transporters was performed with RT-PCR from liver mRNA. Phosphate transporter protein expression was confirmed by Western blotting in basolateral and canalicular LPM and by immunofluorescence in intact liver. Transport studies in canalicular LPM vesicles demonstrated sodium-dependent P(i) uptake. Initial P(i) uptake rates were saturable with increasing P(i) concentrations, exhibiting an apparent K(m) value of approximately 11 muM. P(i) transport was stimulated by an acidic extravesicular pH and by an intravesicular negative membrane potential. These data are compatible with transport characteristics of sodium-phosphate cotransporters NaPi-IIb, PiT-1, and PiT-2, of which the mRNAs were detected in rat liver. On the protein level, NaPi-IIb was detected at the canalicular membrane of hepatocytes and at the brush-border membrane of cholangiocytes. In contrast, PiT-1 and PiT-2 were detected at the basolateral membrane of hepatocytes. We conclude that NaPi-IIb is most probably involved in the reabsorption of P(i) from primary hepatic bile and thus might play an important role in the regulation of biliary P(i) concentration.     

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and beta-mercaptoethanol, with concentrations of 10 mM inhibiting by approximately 40%. DTT's inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [(3)H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.     

Characterization of the transport system for beta-lactam antibiotics and dipeptides in rat renal brush-border membrane vesicles by photoaffinity labeling

The uptake of the alpha-aminocephalosporin cephalexin into brush-border membrane vesicles from rat renal cortex was independent on an inward H+-gradient in contrast to the intestinal transport system. The transport system could be irreversibly inhibited by photoaffinity labeling. Two binding polypeptides for beta-lactam antibiotics and dipeptides with apparent molecular weights 130,000 and 95,000 were identified by photoaffinity labeling with [3H]benzylpenicillin and N-(4-azido[3,5-3H]benzoyl) derivatives of cephalexin and glycyl-L-proline. The uptake of cephalexin and the labeling of the respective binding proteins was inhibited by beta-lactam antibiotics and dipeptides as with intestinal brush-border membranes. These data indicate that the transport systems for beta-lactam antibiotics and dipeptides in the brush-border membrane from rat kidney and small intestine are similar but not identical.     

Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.