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PubMed2003

Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis.

Nucleobases and derivatives like cytokinins and caffeine are translocated in the plant vascular system. Transport studies in cultured Arabidopsis cells indicate that adenine and cytokinin are transported by a common H+-coupled high-affinity purine transport system. Transport properties are similar to that of Arabidopsis purine transporters AtPUP1 and 2. When expressed in yeast, AtPUP1 and 2 mediate energy-dependent high-affinity adenine uptake, whereas AtPUP3 activity was not detectable. Similar to the results from cell cultures, purine permeases (PUP) mediated uptake of adenine can be inhibited by cytokinins, indicating that cytokinins are transport substrates. Direct measurements demonstrate that AtPUP1 is capable of mediating uptake of radiolabeled trans-zeatin. Cytokinin uptake is strongly inhibited by adenine and isopentenyladenine but is poorly inhibited by 6-chloropurine. A number of physiological cytokinins including trans- and cis-zeatin are also efficient competitors for AtPUP2-mediated adenine uptake, suggesting that AtPUP2 is also able to mediate cytokinin transport. Furthermore, AtPUP1 mediates transport of caffeine and ribosylated purine derivatives in yeast. Promoter-reporter gene studies point towards AtPUP1 expression in the epithem of hydathodes and the stigma surface of siliques, suggesting a role in retrieval of cytokinins from xylem sap to prevent loss during guttation. The AtPUP2 promoter drives GUS reporter gene activity in the phloem of Arabidopsis leaves, indicating a role in long-distance transport of adenine and cytokinins. Promoter activity of AtPUP3 was only found in pollen. In summary, three closely related PUPs are differentially expressed in Arabidopsis and at least two PUPs have properties similar to the adenine and cytokinin transport system identified in Arabidopsis cell cultures.

Adenine↗
PubMed2002

Neotrofin is transported out of brain by a saturable mechanism: possible involvement of multidrug resistance and monocarboxylic acid transporters.

Neotrofin (AIT-082; leteprinim potassium) is transported out of brain by a saturable mechanism and in this study the mechanisms mediating this efflux were evaluated. Intracerebroventricular coadministration of [(14)C]Neotrofin with verapamil, a P-glycoprotein inhibitor, probenecid, an organic anion transporter inhibitor, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), a multidrug resistance-associated protein inhibitor, and salicylate or benzoate, both monocarboxylic acid transporter substrates, inhibited the efflux of [(14)C]Neotrofin. Additionally, Neotrofin inhibited the efflux of [(3)H]quinidine from brain. Compounds can diffuse from cerebrospinal fluid (CSF) into extracellular fluid of brain parenchyma and thus, efflux of [(14)C]Neotrofin after intracerebroventricular administration may indicate active transport across choroid plexus epithelium, brain capillary endothelium, or both. To determine whether [(14)C]Neotrofin efflux occurs at the brain capillary endothelium, experiments were performed in which [(14)C]Neotrofin was administered intraparenchymally. The t(1/2) for [(14)C]Neotrofin disappearance from brain after intraparenchymal administration was significantly lower than that for [(3)H]sucrose and the efflux of Neotrofin was inhibited by 600-fold excess of unlabeled Neotrofin, verapamil, MK571, and salicylate. Together, these data suggest that a saturable mechanism for the efflux of Neotrofin is located at the blood-brain barrier and possibly the blood-CSF barrier. It is likely that multiple transporters are involved in the efflux of Neotrofin and these may include multidrug resistance and monocarboxylic acid transporters. These data are discussed in detail with respect to the site of transporter expression, the recent identification of numerous multidrug resistance-associated protein and monocarboxylic acid transporter homologs, the existence of other potential brain efflux transporters, and the availability of specific pharmacological agents with which to distinguish these transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Glucose transport activity in skeletal muscles from transgenic mice overexpressing GLUT1. Increased basal transport is associated with a defective response to diverse stimuli that activate GLUT4.

Glucose transport activity was examined in transgenic mice overexpressing the human GLUT1 glucose transporter in skeletal muscles. Basal transport activity measured in vitro with the glucose analog 2-deoxy-D-glucose (1 mM) was increased 2-8-fold in four different muscle preparations. Incubation of muscles from control nontransgenic littermates with a maximally effective concentration of insulin or with insulin-like growth factor-1 resulted in glucose transport rates that were 2-3-fold higher than basal. In contrast, insulin did not stimulate glucose transport activity in three different muscle preparations from transgenic animals; insulin-like growth factor-1 was similarly ineffective. Activation of System A amino acid transport activity (measured with the nonmetabolizable analog alpha-methylaminoisobutyrate) by insulin was not impaired in muscles from transgenic mice, indicating that the defect does not involve the insulin receptor. In skeletal muscle, glucose transport can be activated by muscle contractions or hypoxia via a pathway separate from that activated by insulin. Incubation of muscles under hypoxic conditions or stimulation of muscles to contract in situ did not increase glucose transport activity in muscles from GLUT1-overexpressing mice, in contrast to the stimulatory effects measured in muscles from control animals. These data suggest that increased glucose flux per se into skeletal muscle results in resistance of GLUT4 to activation by insulin and various other stimuli that activate glucose transport by mechanisms distinct from that of insulin. GLUT1-overexpressing mice thus provide a new model system for studying the effects of glucose-induced resistance to activation of glucose transport.

Amino Acids↗
PubMed1976

Regulation of amino acid transport in growing cells of Streptomyces hydrogenans. I. Modulation of transport capacity and amino acid pool composition during the growth cycle.

(1) The active uptake of different amino acids by growing cells of Streptomyces hydrogenans was shown to be correlated with the physiological age of the cells. During the lag phase of growth the transport capacity increased and attained its highest level when the growth rate was maximum. During further growth the transport capacity declined progressively. The lowest transport activity was observed when the culture shifted into the stationary growth phase. (2) Such modulation of transport capacity was independent on the presence or absence of amino acids in the growth medium of the cells. (3) The size and the composition of the pool of free intracellular amino acids was also undergoing substantial variations during the growth cycle of the culture. In the lag phase, the levels of all amino acids decreased markedly and attained their lowest values at the end of this phase. During further growth the pool size was slowly replenished. (4) Removal of the pool resulted in a considerable gain of transport capacity. Therefore, it was concluded that active amino acid transport in growing Streptomyces hydrogenans is under feedback control by intracellular amino acids. (5) Quantitatively, the modulation of the pool size could not fully account for the variation of the transport capacity. Since a pool-independent stimulation of transport was found to be correlated with the increase of the growth rate of the cells, the possibility is discussed that the stimulation of transport is either due to increased levels of distinct RNA species, which might provide positive feedback signals for transport, or by increased rates of de novo synthesis of transport limiting proteins.

Amino Acids↗
PubMed1990

Na(+)-dependent, active nucleoside transport in mouse spleen lymphocytes, leukemia cells, fibroblasts and macrophages, but not in equivalent human or pig cells; dipyridamole enhances nucleoside salvage by cells with both active and facilitated transport.

Formycin B influx studies have shown that P388 and L1210 mouse leukemia cells, mouse L929 cells, mouse RAW 309 Cr.1 cells, LK35.2 mouse B-cell hybridoma cells and cultured mouse peritoneal macrophages express both Na(+)-dependent, active and nonconcentrative, facilitated nucleoside transport systems. In the mouse cell lines, active transport represented only a minor nucleoside transport component and was detected only by measuring formycin B uptake in the presence of dipyridamole or nitrobenzylthioinosine, strong inhibitors of facilitated, but not of active, nucleoside transport. Inhibition of facilitated transport resulted in the concentrative accumulation of formycin B in cells expressing active nucleoside transport. Concentrative formycin B accumulation was abolished by treatment of the cells with gramicidin or absence of Na+ in the extracellular medium and strongly inhibited by ATP depletion or ouabain treatment. Mouse macrophages accumulated formycin B to 70-times the extracellular concentration in the absence of dipyridamole during 90 min of incubation at 37 degrees C. Thus active transport represents a major nucleoside transport system of these cells, similarly as previously reported for mouse spleen lymphocytes. In contrast to the various types of mouse cells, active formycin B transport was not detected in human HeLa cells, human H9, Jurkat and CEM T lymphoidal cells and pig spleen lymphocytes. These cells expressed only facilitated nucleoside transport with kinetic properties similar to those of the facilitated transporters of other mammalian cells.

Animals↗
PubMed1989

Hybridization analysis of RNA transported from rat liver nuclei in response to 35 kDa normal and 60 kDa messenger RNA transport factors.

The transport of messenger RNA (mRNA) in response to normal adult (35 kDa) and oncofetal (60 kDa) transport factors has been studied in a reconstituted cell-free system. Poly(A)+ mRNA sequences transported by the 35 kDa and 60 kDa transport factors were compared by cDNA:RNA hybridization kinetics. Heterologous hybridization reactions indicated that a proportion of messengers transported in response to the 35 kDa factor were absent or at a markedly reduced abundance in the mRNA released by the 60 kDa factor. Recombinant DNA probes containing cDNA inserts were used to quantitate transport of rat-liver-specific alpha 2 mu-globulin and albumin mRNA from isolated nuclei in presence of the normal and tumor-specific transport factors. More alpha 2 mu-globulin and albumin messenger sequences were transported in response to the 35 kDa transport factor as compared to the 60 kDa factor. These results indicate that the 35 kDa transport protein isolated from rat liver cytosol and the 60 kDa transport protein isolated from hepatoma cytosol, differ significantly in specificity for the classes of RNA sequences released from nuclei. Monoclonal antibodies against the 60 kDa factor do not cross-react with the 35 kDa factor or other proteins as determined by the immunobioassay and by the Western blot technique.

Albumins↗
PubMed2004

Life support for trauma and transport: a mobile ICU for safe in-hospital transport of critically injured patients.

BACKGROUND: In-hospital transport of newly injured patients is complicated by inadequate monitoring and adverse events. LSTAT (Life Support for Trauma and Transport, Integrated Medical Systems Inc) is a platform with multiple integrated systems (ventilator, defibrillator, suction, hemodynamic monitors, infusion and invasive monitoring channels, capnography, blood analysis, and electrocardiography) that allow seamless monitoring and effective life-saving interventions during transport. The platform functions as a mobile ICU and has preliminarily been tested with success in combat settings. This is the first evaluation of LSTAT in the civilian transport arena. STUDY DESIGN: Major trauma patients requiring trauma team activation, who were transported from the Emergency Department through different hospital departments (usually CT or angiography) to the ICU or operating room were included prospectively (December 2002 through April 2003). Patients were monitored and transported either by conventional means (conventional group) or by LSTAT (LSTAT group). Primary outcomes related to resource consumption and process of care; secondary outcomes related to clinical events. A questionnaire was completed by the surgeons participating in transports to document perceptions and preferences about means of in-hospital transport. RESULTS: Of 178 patients enrolled, 85 (48%) were in the LSTAT and 93 (52%) in the conventional groups. The two groups were similar except for age and mechanism of injury. Time of hand-bagging, preparation for transport, and return of blood results was significantly shorter in the LSTAT than in the conventional group (p < 0.001 for all). Significantly fewer LSTAT than conventional transports required more than one escorting physician (p < 0.001). Significantly more surveyed surgeons preferred LSTAT to conventional methods to transfer patients. There were no differences in adverse events, hospital stay, or mortality between the two groups. CONCLUSIONS: LSTAT emerges as a safe and convenient method of in-hospital transport. It allows uninterrupted monitoring, immediate response to physiologic changes, and reduction in human resource consumption. Process of care is improved. LSTAT's potential to improve clinical outcomes needs to be tested in different environments, including the prehospital setting.

Adolescent↗
PubMed1980

Perinatal transport to a regional perinatal center in a metropolitan area: Maternal versus neonatal transport.

A prospective study was undertaken to evaluate antenatal maternal referral, acute maternal transport, and neonatal transport to a regional perinatal center in a metropolitan area. During an 18-month period, there were 143 antenatal maternal referrals, 254 antenatal maternal transports, and 506 neonatal transports. Indications for the antenatal referrals were maternal diseases of a chronic nature. This group had a 28.7% incidence of delivery by primary cesarean section, a 15.8% incidence of low-birth weight infants, and a 30.8% incidence of neonatal admissions to the Neonatal Intensive Care Unit (NICU). The perinatal mortality rate was 13.7 per thousand which compares favorably with that of low-risk obstetric patients. Indications for the antenatal maternal transports were mainly premature labor and/or premature rupture of the membranes and third-trimester bleeding. There was a 37% incidence of delivery by primary cesarean section and a 71.5% incidence of low-birth weight infants, and 77.4% of the newborn infants were admitted to the NICU. The perinatal mortality rate was 137.4 per thousand live births. Compared to postnatal neonatal transports, the neonates transported antenatally had a slightly higher mortality rate which was not statistically significant. One hundred thirty-one neonates transported antenatally could be matched in sequential order of admission with 131 neonates transported postnatally of comparable birth weights and gestational ages. Although there was a significant increase in the incidence of delivery by cesarean section among the mothers transported antenatally, the neonates in this group had a significantly lower incidence of respiratory distress syndrome and other morbidity and a shorter hospitalization. This study suggests that antenatal referral and transport of high-risk parturient patients to a regional perinatal center may significantly decrease neonatal morbidity and length of hospitalization.

California↗
PubMed1998

Aluminum transport out of brain extracellular fluid is proton dependent and inhibited by mersalyl acid, suggesting mediation by the monocarboxylate transporter (MCT1).

Blood brain barrier transport of aluminum citrate was assessed in rats by microdialysis of the jugular vein as well as the right and left frontal cortices. Previous studies (Allen et al., 1995. Evidence for energy-dependent transport of aluminum out of brain extracellular fluid. Toxicology 92, 193-202; Ackley and Yokel, 1997. Aluminum citrate is transported from brain into blood via the monocarboxylic acid transporter located at the blood-brain barrier. Toxicology 120, 89-97), and the current study, demonstrated that the steady-state brain-to-blood ratio of the unbound extracellular aluminum immediately surrounding the microdialysis probe is less than 1, suggesting the presence of a process other than diffusion across the blood brain barrier. It was speculated that a monocarboxylate transporter at the blood brain barrier was maintaining this ratio at less than 1 (Ackley and Yokel, 1997). Monocarboxylate transporters are proton co-transporters. Decreasing extracellular pH (increasing proton availability) increases monocarboxylate transport. After alkalinizing the dialysate perfusing a brain microdialysis probe (to pH = 10.2), the steady-state aluminum brain-to-blood ratio increased from 0.35 to 0.80. The addition of the proton ionophore, p-(trifluoromethoxy)phenylhydrazone (FCCP) (1 mM), to brain dialysate increased this ratio from 0.21 to 0.61. These increased ratios suggest that a proton-dependent process is removing Al from brain extracellular fluid. The monocarboxylate transporter is the only known proton-dependent transporter at the blood-brain barrier. There are two known isoforms of this transporter in the rodent, MCT1 and MCT2. Organomercurial thiol reagents, such as mersalyl acid, inhibit MCT1 but not MCT2. Mersalyl acid (50 mM) addition to brain dialysate increased the steady-state aluminum brain-to-blood ratio from 0.19 to 0.87, suggesting that MCT1 is at least partially mediating the efflux of aluminum from brain extracellular fluid.

Aluminum↗
PubMed2003

Changes in adenosine transport associated with melaminophenyl arsenical (Mel CY) resistance in Trypanosoma evansi: down-regulation and affinity changes of the P2 transporter.

Studies of the kinetics of adenosine transport were carried out on the P1 and P2 transporters of drug-sensitive Trypanosoma evansi and its cloned derivative, resistant to the melaminophenyl arsenical Mel CY. Characterization of adenosine uptake was made by estimation of the maximum concentration taken up at time infinity (Amax). Amax on the P2 transporter of sensitive T. evansi was greater than Amax on the P1 transporter. Amax of the P2 transporter was significantly decreased in drug-resistant trypanosomes. The effect of adenosine concentration and inhibitors, on the rate of adenosine uptake, was described by Michaelis-Menten equations. In sensitive T. evansi, the maximum velocity of adenosine uptake (Vmax) of the P2 transporter was 2-fold greater than Vmax of the P1 transporter. The Vmax of the P2 transporter in resistant parasites was reduced 9-fold. The binding constants Km and Ki on the P2 transporter of resistant T. evansi, showed that resistance was associated with an increased affinity for adenosine, and a decreased affinity for adenine and Berenil. We suggest that resistance to melaminophenyl arsenicals in T. evansi, occurs via 2 mechanisms: (1) a reduction in the number of expressed P2 transporter molecules resulting in decreased uptake of melaminophenyl arsenicals; (2) a change in the binding properties of the P2 transporter.

Adenine↗
PubMedDate not supplied

Prehospital transport time and mode of transportation in Zenica, Bosnia.

STUDY OBJECTIVES: To provide a descriptive analysis of the prehospital emergency medical transportation system operating in and around a major Bosnian city, and to discuss the effect of the war on that transportation system. DESIGN: A prospective, consecutive sample study. SETTING: The largest tertiary-care referral hospital currently operating in central Bosnia. PARTICIPANTS: 158 consecutive patients who sought care in the trauma and medical receiving rooms at Zenica Hospital. METHOD AND MEASUREMENTS: Patients were surveyed as to time of transportation to the site of first medical care and to Zenica Hospital, and modes of transportation used. RESULTS: Time to transport to first care within the city of Zenica is 30 +/- 21 minutes. Time to transport to Zenica Hospital from within Zenica is 51 +/- 39 minutes. Time to transport to first care outside of Zenica is 77 +/- 56 minutes. Time to transport to Zenica Hospital from outside the city of Zenica is 178 +/- 94 minutes. The prehospital emergency medical service was used in 11.7% of cases reviewed. CONCLUSION: The majority of patients with major injury and illness are unable to obtain prehospital transportation and medical care through informal modes of transportation. The existing prehospital emergency medical services system is inadequate for the numbers of patients requiring such services, and transportation time from outside the city of study is especially prolonged.

Ambulances↗
PubMed1989

Analysis of protein-mediated 3-O-methylglucose transport in rat erythrocytes: rejection of the alternating conformation carrier model for sugar transport.

3-O-Methylglucose (3OMG) transport in rat erythrocytes (RBCs) is mediated by a low-capacity, facilitated diffusion-type process. This study examines whether the characteristics of sugar transport in rat RBCs are consistent with the predictions of two diametric, theoretical mechanisms for sugar transport. The one-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites are mutually exclusive. The two-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites can exist simultaneously but may interact in a cooperative fashion when occupied by substrate. Michaelis and velocity parameters for saturable 3OMG transport in rat erythrocytes at 24 degrees C were obtained from initial rate measurements of 3OMG transport. The results are incompatible with the predictions of the one-site carrier but are consistent with the predictions of a symmetric two-site carrier, displaying negligible cooperativity between substrate binding sites. This allows reduction of the two-site carrier transport equations to a form containing fewer constants than the one-site carrier equations without limiting their predictive success. While the available evidence does not prove that rat erythrocyte sugar transport is mediated by a two-site mechanism, we conclude that adoption of the formally more complex one-site model for sugar transport in rat erythrocytes is unnecessary and unwarranted. Counterflow experiments have also been performed in which the time course of radiolabeled 3OMG uptake is measured in cells containing saturating levels of 3OMG. The results of these experiments are consistent with the hypothesis [Naftalin et al. (1985) Biochim. Biophys. Acta 820, 235-249] that exchange of sugar between intracellular compartments (cell water and hemoglobin) can be rate limiting for transport under certain conditions.

3-O-Methylglucose↗
PubMed1997

Sequence alignments of the H(+)-dependent oligopeptide transporter family PTR: inferences on structure and function of the intestinal PET1 transporter.

PURPOSE: To study the structure and function of the intestinal H+/ peptide transporter PET1, we compared its amino acid sequence with those of related transporters belonging to the oligopeptide transporter family PTR, and with more distant transporter families. METHODS: We have developed a new approach to the sequence analysis of proteins with multiple transmembrane domains (TMDs) which takes into account the repeated TMD-loop topology. In addition to conventional analyses of the entire sequence, each TMD and its adjacent loop residues (= TMD segments) were analyzed separately as independent structural units. In combination with hydropathy analysis, this approach reveals any changes in the order of the TMD segments in the primary structure and permits TMD alignments among divergent structures even if rearrangements of the order of TMD segments have occurred in the course of evolution. RESULTS: Alignments of TMD segments indicate that the TMD order in PTR transporters may have changed in the process of evolution. Consideration of such changes permits the alignment of homologous TMD segments from PTR transporters belonging to distant akaryotic and eukaryotic phyla. Multiple alignments of TMDs reveal several highly conserved regions that may play a role in transporter function. In comparing the PTR transporters with other transporter gene families, alignment scores using the entire primary structure are too low to support a finding of probable homology. However, statistically significant alignments were observed among individual TMD segments if one disregards the order in which they occur in the primary structure. CONCLUSIONS: Our results support the hypothesis that the PTR transporters may have evolved by rearrangement, duplication, or insertions and deletions of TMD segments as independent modules. This modular structure suggests new alignment strategies for determining functional domains and testing relationship among distant transporter families.

Amino Acid Sequence↗
PubMed1984

Renal transport of neutral amino acids. Tubular localization of Na+-dependent phenylalanine- and glucose-transport systems.

The transport properties for phenylalanine and glucose in luminal-membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit kidney were studied by a spectrophotometric method. Uptake of phenylalanine as well as of glucose by the two types of membrane vesicles was found to be Na+-dependent, electrogenic and stereospecific. Na+-dependent transport of L-phenylalanine by outer-cortical membrane vesicles could be accounted for by one transport system (KA congruent to 1.5 mM). By contrast, in the outer-medullary preparation, L-phenylalanine transport occurred via two transport systems, namely a high-affinity system with K1A congruent to 0.33 mM and a low-affinity system with K2A congruent to 7 mM respectively. Na+-dependent uptake of D-glucose by pars convoluta and pars recta membrane vesicles could be described by single, but different, transport systems, namely a low-affinity system with KA congruent to 3.5 mM and a high-affinity system with KA congruent to 0.30 mM respectively. Attempts to calculate the stoichiometry of the different Na+/D-glucose transport systems by using Hill-type plots revealed that the ratio of the Na+/hexose co-transport probably is 1:1 in the case of pars convoluta and 2:1 in membrane vesicles from pars recta. The Na+/L-phenylalanine stoichiometry of the pars convoluta transporter probably is 1:1. Both the high-affinity and the low-affinity Na+-dependent L-phenylalanine transport system of pars recta membrane vesicles seem to operate with a 1:1 stoichiometry. The physiological importance of the arrangement of low-affinity and high-affinity transport systems along the kidney proximal tubule is discussed.

Animals↗
PubMed1986

Biochemical and functional characterization of the rat liver glucose-transport system. Comparisons with the adipocyte glucose-transport system.

The properties of the glucose-transport systems in rat adipocytes and hepatocytes were compared in cells prepared from the same animals. Hormones and other agents which cause a large stimulation of 3-O-methylglucose transport in adipocytes were without acute effect in hepatocytes. Hepatocytes displayed a lower affinity for 3-O-methylglucose (20 mM) and alternative substrates than adipocytes (6 mM), whereas inhibitor affinities were similar in both cell types. The concentration and distribution of glucose transporters were determined by Scatchard analysis of D-glucose-inhibitable [3H]cytochalasin B binding to subcellular fractions. In liver, most of the transporters were located in the plasma membrane (42 +/- 5 pmol/mg of protein) with a small amount (4 +/- 3 pmol/mg) in the low-density microsomal fraction ('microsomes'), the reverse of the situation in adipocytes. Glucose transporters were covalently labelled with [3H]cytochalasin B by using the photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate and analysed by SDS/polyacrylamide-gel electrophoresis. A single D-glucose-inhibitable peak with a molecular mass of 40-50 kDa was seen in both plasma membrane and low-density microsomes. This peak was further characterized by isoelectric focusing and revealed a single peak of specific [3H]cytochalasin B binding at pI 6.05 in both low-density microsomes and plasma membrane, compared with peaks at pI 6.4 and 5.6 in adipocyte membranes. In summary: the glucose-transport system in hepatocytes has a lower affinity and higher capacity than that in adipocytes, and is also not accurately modulated by insulin; the subcellular distribution of glucose transporters in the liver suggests that few intracellular transporters would be available for translocation; the liver transporter has a molecular mass similar to that of the adipocyte transporter; the liver glucose transporter exists as a single charged form (pI 6.05), compared with the multiple forms in adipocytes. This difference in charge could reflect a functionally important difference in molecular structure between the two cell types.

3-O-Methylglucose↗
PubMed1998

Cloning and functional characterization of a Brassica napus transporter that is able to transport nitrate and histidine.

A full-length cDNA for a membrane transporter was isolated from Brassica napus by its sequence homology to a previously cloned Arabidopsis low affinity nitrate transporter. The cDNA encodes a predicted protein of 589 amino acid residues with 12 putative transmembrane domains. The transporter belongs to a multigene family with members that have been identified in bacteria, fungi, plants, and animals and that are able to transport a range of different nitrogen-containing substrates, including amino acids, peptides, and nitrate. To identify the substrates of this plant gene, we have expressed the protein in Xenopus oocytes. The properties of the transporter are consistent with a proton cotransport mechanism for nitrate, and the voltage dependence of the Km for nitrate was determined. The Km for nitrate was shown to increase from 4 to 14 mM as the membrane voltage became more negative from -40 to -180 mV. Oocytes expressing the gene could accumulate internal nitrate to concentrations higher than those measured in water-injected controls. A range of different substrate molecules for the transporter was tested, but of these, histidine gave the largest currents, although the affinity was in the millimolar range. The pH dependence of the activity of the transporter was different for the substrates, with histidine transport favored at alkaline and nitrate at acid external pH. Kinetic analysis of the mechanism of histidine transport suggests a cotransport of protons and the neutral form of the amino acid, with the Km for histidine decreasing at more negative membrane voltages. This gene is the first member of this family of transporters for which the transport of two very different types of substrate, nitrate and histidine, has been demonstrated.

Animals↗
PubMed2002

Identification of a novel Na+-independent acidic amino acid transporter with structural similarity to the member of a heterodimeric amino acid transporter family associated with unknown heavy chains.

We identified a novel Na(+)-independent acidic amino acid transporter designated AGT1 (aspartate/glutamate transporter 1). AGT1 exhibits the highest sequence similarity (48% identity) to the Na(+)-independent small neutral amino acid transporter Asc (asc-type amino acid transporter)-2 a member of the heterodimeric amino acid transporter family presumed to be associated with unknown heavy chains (Chairoungdua, A., Kanai, Y., Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001) J. Biol. Chem. 276, 49390-49399). The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits of the heterodimeric amino acid transporter family is conserved for AGT1. Because AGT1 solely expressed or coexpressed with already known heavy chain 4F2hc (4F2 heavy chain) or rBAT (related to b(0,+)-amino acid transporter) did not induce functional activity, we generated fusion proteins in which AGT1 was connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the Na(+)-independent transport activity for acidic amino acids. Distinct from the Na(+)-independent cystine/glutamate transporter xCT structurally related to AGT1, AGT1 did not accept cystine, homocysteate, and l-alpha-aminoadipate and exhibited high affinity to aspartate as well as glutamate, suggesting that the negative charge recognition site in the side chain-binding site of AGT1 would be closer to the alpha-carbon binding site compared with that of xCT. The AGT1 message was predominantly expressed in kidney. In mouse kidney, AGT1 protein was present in the basolateral membrane of the proximal straight tubules and distal convoluted tubules. In the Western blot analysis, AGT1 was detected as a high molecular mass band in the nonreducing condition, whereas the band shifted to a 40-kDa band corresponding to the AGT1 monomer in the reducing condition, suggesting the association of AGT1 with other protein via a disulfide bond. The finding of AGT1 and Asc-2 has established a new subgroup of the heterodimeric amino acid transporter family whose members associate not with 4F2hc or rBAT but with other unknown heavy chains.

Amino Acid Sequence↗
PubMed1999

Indispensability of transmembrane domains of Golgi UDP-galactose transporter as revealed by analysis of genetic defects in UDP-galactose transporter-deficient murine had-1 mutant cell lines and construction of deletion mutants.

UDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galactosyltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters. We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations. Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein.

Amino Acid Sequence↗