Effect of lithium ion on melibiose transport inEscherichia coli

Summary Both Li⁺ and Na⁺ stimulated the uptake of thiomethylgalactoside by the melibiose transport system ofEscherichia coli. On the other hand, Li⁺ inhibited the growth of cells on melibiose as a sole source of carbon. This inhibition was specific for melibiose, and Li⁺ had no effect on growth of cells on glucose, galactose, lactose, or glycerol. The effect of the cation on melibiose transport was investigated in a mutant which cannot utilize glucose. After entry into this cell, melibiose is cleaved into glucose and galactose by -galactosidase, and the resulting glucose is excreted. Since the entry step was found to be rate-limiting, glucose production could be taken as a measure of melibiose transport. Li⁺ inhibited the transport of melibiose, but not the induction of the melibiose operon nor the activity of -galactosidase. Li⁺ was found to inhibit the entry ofp-nitrophenyl--d-galactoside, but notp-nitrophenyl--d-galactoside entry. Thus, the cation specificity for the melibiose membrane carrier varies with different transport substrates.     

Cation-Sugar Cotransport in the Melibiose Transport System of Escherichia coli

The entry of Na⁺ or H⁺ into cells of Escherichia coli via the melibiose transport system was stimulated by the addition of certain galactosides. The principal cell used in these studies (W3133) was a lactose transport negative strain of E. coli possessing an inducible melibiose transport system. Such cells were grown in the presence of melibiose, washed, and incubated in the presence of 25 μM Na⁺. The addition of thiomethylgalactoside (TMG) resulted in a fall in Na⁺ concentration in the incubation medium. No TMG-stimulated Na⁺ movement was observed in uninduced cells. In an α-galactosidase negative derivative of W3133 (RA11) a sugar-stimulated Na⁺ uptake was observed in meliboise-induced cells on the addition of melibiose, thiodigalactoside, methyl-α-galactoside, methyl-β-galactoside, and galactose, but not lactose. It was inferred from these studies that the substrates of the melibiose system enter the cell on the melibiose carrier associated with the simultaneous entry of Na⁺ when this cation is present in the incubation medium.

Extracellular pH was measured in unbuffered suspensions of induced cells in order to study proton movement across the membrane of cells exposed to different galactosides. In the absence of external Na⁺ or Li⁺ the addition of melibiose or methyl-α-galactoside resulted in marked alkalinization of the external medium (consistent with H⁺-sugar cotransport). On the other hand TMG, thiodigalactoside, and methyl-β-galactoside gave no proton movement under these conditions. When Na⁺ was present, the addition of TMG or melibiose resulted in acidification of the medium. This observation is consistent with the view that the entry of Na⁺ with TMG or melibiose carries into the cell a positive charge (Na⁺) which provides the driving force for the diffusion of protons out of the cell. It is concluded that the melibiose carrier recognition of cations differs with different substrates.     

Bacterial transporters: Charge translocation and mechanism

A comparative review of the electrophysiological characterization of selected secondary active transporters from Escherichia coli is presented. In melibiose permease MelB and the Na⁺/proline carrier PutP pre-steady-state charge displacements can be assigned to an electrogenic conformational transition associated with the substrate release process. In both transporters cytoplasmic release of the sugar or the amino acid as well as release of the coupling cation are associated with a charge displacement. This suggests a common transport mechanism for both transporters. In the NhaA Na⁺/H⁺ exchanger charge translocation due to its steady-state transport activity is observed. A new model is proposed for pH regulation of NhaA that is based on coupled Na⁺ and H⁺ equilibrium binding.     

Spontaneous and reversible high-frequency frameshifts originating a phase transition in the carotenoid biosynthesis pathway of the phototrophic bacterium Rhodobacter sphaeroides 2.4.1

Summary The nucleotide sequence of the melB gene coding for the Na⁺(Li⁺)/melibiose symporter of Salmonella typhimurium LT2 was determined, and its amino acid sequence was deduced. It consists of 1428 bp, corresponding to a protein of 476 amino acid residues (calculated molecular weight 52800). The amino acid sequence is homologous to that of the melibiose permease of Escherichia coli K12, with 85% identical residues. All, except one, of the amino acid residues that have been reported to be important for cation or substrate recognition in the melibiose permease of E. coli are conserved in the melibiose permease of S. typhimurium. In addition, part of the sequence resembles the lactose permease of Streptococcus thermophilus, the animal glucose transporter (GLUT1), the plasmid-coded raffinose permease (RafB), and the NADH-ubiquinone oxidoreductase chain 4 (Nuo4) of Aspergillus amstelodami.The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank/EMBL Data Bank with accession number X62101     

Characterization of a Na+/glucose cotransporter cloned from rabbit small intestine

Summary The Na⁺/glucose cotransporter from rabbit intestinal brush border membranes has been cloned, sequenced, and expressed inXenopus oocytes. Injection of cloned RNA into oocytes increased Na⁺/sugar cotransport by three orders of magnitude. In this study, we have compared and contrasted the transport properties of this cloned protein expressed inXenopus oocytes with the native transporter present in rabbit intestinal brush borders. Initial rates of¹⁴C--methyl-d-glucopyranoside uptake into brush border membrane vesicles andXenopus oocytes were measured as a function of the external sodium, sugar, and phlorizin concentrations. Sugar uptake into oocytes and brush borders was Na⁺-dependent (Hill coefficient 1.5 and 1.7), phlorizin inhibitable (K _i 6 and 9 m), and saturable (-methyl-d-glucopyranosideK _m 110 and 570 m). The sugar specificity was examined by competition experiments, and in both cases the selectivity wasd-glucose>-methyl-d-glucopyranoside>d-galactose>3-O-methyl-d-glucoside. In view of the close similarity between the properties of the cloned protein expressed in oocytes and the native brush border transporter, we conclude that we have cloned the classical Na⁺/glucose cotransporter.     

Membrane transport ofp-nitrophenyl-α-galactoside by the melibiose carrier ofEscherichia coli

Summary p-Nitrophenyl--galactoside (-pNPG) was found to be a substrate for the melibiose transport system ofEscherichia coli. This sugar enters induced cells via the carrier and is split by -galactosidase to galactose andp-nitrophenol. In mutant cells lacking the -galactosidase [³H]--pNPG accumulated to concentrations 15 times higher than the external medium. The transport of -pNPG is inhibited by both Na⁺ and Li⁺. Na⁺ (10mm) reduced the K_m for -pNPG from 0.45 to 0.18mm and reduced theV _max from 6.7 nmoles/min/mg dry wt to a value of 3.0.     

Co-transport of Na+ and methul-beta-D-thiogalactopyranoside mediated by the melibiose transport system of Escherichia coli.

Na⁺-dependent transport of methyl-β-D-thiogalactopyranoside (TMG) mediated by the melibiose transport system was investigated in $\text{Escherichia coli}$ mutants lacking the lactose transport system. When an inwardly-directed electro-chemical potential difference of Na⁺ was imposed across the membrane of energy depleted cells, transient uptake of TMG was observed. Addition of TMG to cell suspensions under anaerobic conditions caused a transient acidification of the medium. This acidification was observed only in the presence of Na⁺ or Li⁺. These results support the idea that TMG is taken up by a mechanism of Na⁺-TMG co-transport via the melibiose transport system in $\text{Escherichia coli}$.     

Studies on the kinetics of Na+/H+ exchange in OK cells: Introduction of a new device for the analysis of polarized transport in cultured epithelia

Summary The present study describes a new perfusion technique—based on the use of a routine spectrofluorometer—which enables fluorometric evaluation of polarity, regulation and kinetics of Na⁺/H⁺ exchange at the level of an intact monolayer. Na⁺/ H⁺ exchange was evaluated in bicarbonate-free solutions in OK (opossum kidney) cells, a renal epithelial cell line. Na⁺/H⁺ exchange activity was measured by monitoring changes in intracellular pH (pH _i ) after an acid load, using the pH-sensitive dye 27-bis (carboxyethyl) 5–6-carboxy-fluorescein (BCECF). Initial experiments indicated that OK cells grown on a permeable support had access to apical and basolateral perfusion media. They also demonstrate that OK cells express an apical pH _i , recovery mechanism, which is Na⁺ dependent, ethylisopropylamiloride (EIPA) sensitive and regulated by PTH. Compared to resting conditions (pH _i =7.68; pH _o =7.4) where Na⁺/H⁺ exchange is not detectable, transport rate increased as pH _i decreased. A positive cooperativity characterized the interaction of internal H⁺ with the exchanger, and suggests multiple H⁺ binding sites. In contrast, extracellular [Na⁺] increased transport with simple Michaelis-Menten kinetics. The apparent affinity of the exchanger for Na⁺ was 19mM at an intracellular pH of 7.1 and 60mM at an intracellular pH of 6.6. Inhibition of Na⁺/H⁺ exchange activity by EIPA was competitive with respect to extracellular [Na⁺] and theK _i was 3.4 M. In conclusion, the technique used in the present study is well suited for determination of mechanisms involved in control of epithelial cell pH _i and processes associated with their polarized expression and regulation.     

Cysteine substitutions for individual residues in helix VI of the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a cytoplasmic membrane protein that mediates the cotransport of galactosides with H⁺, Na⁺, or Li⁺. In this study we used cysteine-scanning mutagenesis to try to gain information about the position of transmembrane helix VI in the three-dimensional structure of the melibiose carrier. We constructed 23 individual cysteine substitutions in helix VI and an adjacent loop of the carrier. The resulting melibiose carriers retained 22–100% of their ability to transport melibiose. We tested the effect of the hydrophilic sulfhydryl reagent p-chloromercuri-benzenesulfonic acid (PCMBS) on the cysteine-substitution mutants and we found that there was no inhibition of melibiose transport in any of the mutants. We suggest that helix VI is imbedded in phospholipid and does not face the aqueous channel through which melibiose passes. Received: 6 March 2001/Revised: 14 May 2001     

Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick

Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H⁺ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K⁺ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H⁺ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na⁺/H⁺ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na⁺ gradient. The kinetics of the Na⁺/H⁺ exchange reaction at pH 6.0 [K _t for Na⁺=57mm,V _max=42 mmol H⁺/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K _t for Na⁺=15mm,V _max=1.7 mmol H⁺/liter 3OMG space/min). Experiments involving imposed K⁺ gradients suggest that these cells have negligible K⁺/H⁺ exchange capability. They exhibit limited but measurable H⁺ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate.     

Amiloride-sensitive Na+ transport in human red cells: Evidence for a Na/H exchange system

Summary The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na⁺ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 m) and methazolamide (400 m). Red cells with different internal pH (pH _i =6.4, 7.0 and 7.8) were prepared and Na⁺ influx was measured at different external pH (pH _o =6.0, 7.0, 8.0). Na⁺ influx into acid-loaded cells (pH _i =6.4) markedly increased when pH _o was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na⁺/H⁺ exchange systems blocked about 60% of the H⁺-induced Na⁺ entry, while showing small inhibitory effects in the absence of pH gradients. When pH₀ was kept at 8.0, the amiloride-sensitive Na⁺ entry was abolished as pH _i was increased from 6.4 to 7.8. Moreover, measurements of H⁺ efflux into lightly buffered media indicated that the imposition of an inward Na⁺ gradient stimulated a net H⁺ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na⁺ (Na _i ⁺ =Na ₀ ⁺ =15mm,Em=+6.7 mV), an outward H⁺ gradient (pH _i =6.4, pH₀=8.0) promoted a net amiloride-sensitive Na⁺ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na⁺/H⁺ exchange system in human red cells.     

Sodiumd-glucose cotransport in the gill of marine mussels: Studies with intact tissue and brush-border membrane vesicles

Summary Glucose transport was studied in marine mussels of the genusMytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na⁺-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na⁺-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na⁺, and concentrative glucose transport was observed in the presence of an inwardly directed Na⁺ gradient. There was a single saturable pathway for glucose uptake, with an apparentK _t of 3 m in BBMV and 9 m in intact gills. The kinetics of Na⁺ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na⁺ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with aK _i of 44 m. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na⁺-glucose cotransporters in other systems;d-glucose and -methyl glucopyranoside were the most effective inhibitors of Na⁺-glucose transport,d-galactose was intermediate in its inhibition, and there was little or no effect ofl-glucose,d-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na⁺-glucose uptake, with an apparentK _i of 154nm in BBMV and 21nm in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.     

Protein kinase C activates the renal apical membrane Na+/H+ exchanger

Summary Studies were performed on purified brush-border membranes from the kidney of the rabbit to examine the relation between protein kinase C and the Na⁺/H⁺ exchanger in these membranes. The brush-border membranes were transiently opened by exposure to hypotonic media and the membrane proteins phosphorylated by exposure to ATP and phorbol esters or partially purified protein kinase C. The membranes were resealed and the intravesicular space acidified by incubation in a sodium-free isotonic solution (pH 5.5). The rate of uptake of 1mm ²²Na⁺ (pH 7.5), with and without amiloride (1mm), was assayed and the proton gradient-stimulated, amiloride-inhibitable component of²²Na⁺ taken as a measure of the activity of the Na⁺/H⁺ exchanger. 12-0-tetradecanoyl phorbol-13-acetate (TPA) increased the amiloride-sensitive component of²²Na⁺ uptake TPA did not affect the amiloride-insensitive component of²²Na⁺ uptake or the equilibrium concentration of sodium. TPA also did not affect the rate of dissipation of the proton gradient in the absence of sodium or the rate of sodium-dependent or-independent uptake ofd-glucose. Other active phorbol esters stimulated the rate of Na⁺/H⁺ exchange, but phorbol esters of the 4 configuration did not. Incubation of the opened membranes in partially purified protein kinase C increased the rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake. The stimulatory effect of TPA and protein kinase C was not additive. In the absence of ATP, neither TPA nor protein kinase C affected Na⁺/H⁺ exchange transport. To determine the membrane-bound protein substrates, parallel experiments were conducted with -[³²P] ATP in the phosphorylating solutions. The reaction was stopped by SDS and the phosphoproteins resolved by PAGE and autoradiography. TPA stimulation of protein kinase C resulted in phosphorylation of approximately 13 membrane-bound proteins ranging in apparent molecule from 15,000 to 140,000 daltons. These studies indicate that activation of endogenous renal brush-border protein kinase C by phorbol esters or exposure of these membranes to exogenous protein kinase C increases the rate of proton gradient-stimulated, amiloride-inhibitable sodium transport. Protein kinase C activation also results in phosphorylation of a finite number of membrane-bound proteins.     

Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

Summary We have investigated the kinetic properties of the human red blood cell Na⁺/H⁺ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H⁺ (H _i ) and external Na⁺ (Na _o ) in red blood cells of normal subjects.Red blood cells with different cell Na⁺ (Na _i ) and pH (pH _i ) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na⁺ influx were measured by varying pH _i (from 5.7 to 7.4), external pH (pH _o ), Na _i and Na _o and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na⁺ influx (Na _i <2.0 mmol/liter cell, Na _o = 150mm) increased sigmoidally (Hill coefficient 2.5) when pH _i fell below 7.0 and the external pH _o was 8.0, but increased linearly at pH _o 6.0. The net Na⁺ influx driven by an outward H⁺ gradient was estimated from the difference of Na⁺ influx at the two pH _o levels (pH _o 8 and pH _o 6). The H⁺-driven Na⁺ influx reached saturation between pH _i 5.9 and 6.1. TheV _max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK _m for H _i to activate H⁺-driven Na⁺ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na⁺/H⁺ exchange. The stoichiometric ratio between H⁺-driven Na⁺ influx and Na⁺-driven H⁺ efflux was 11. The dependence of Na⁺ influx from Na _o was studied at pH _i 6.0, and Na _i lower than 2 mmol/liter cell at pH _o 6.0 and 8.0. The meanK _m for Na _o of the H⁺-gradient-driven Na⁺ influx was 55±7mm.An increase in Na _i from 2 to 20 mmol/liter cell did not change significantly H⁺-driven net Na⁺ influx as estimated from the difference between unidirectional²²Na influx and efflux. Na⁺/Na⁺ exchange was negligible in acid-loaded, DIDS-treated cells. Na⁺ and H⁺ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na⁺ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na⁺/H⁺ exchanger.It is concluded that human red cell Na⁺/H⁺ exchange performs 11 exchange of external Na⁺ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H⁺ and external Na⁺ is similar to other cells, but it displays a larger variability in theV _max between individuals.     

Anaerobic growth of Salmonella typhimurium on l(+)- and d(−)-tartrate involves an oxaloacetate decarboxylase Na+ pump

We show here that the Enterobacterium Salmonella typhimurium LT2 has the capacity to grow anaerobically on l(+)- or d(-)-tartrate as sole carbon and energy source. Growth on these substrates was Na⁺-dependent and involved the l(+)- or d(-)-tartrate-inducible expression of oxaloacetate decarboxylase. The induced decarboxylase was closely related to the oxaloacetate decarboxylase Na⁺ pump of Klebsiella pneumoniae as shown by the sensitivity towards avidin, the location in the cytoplasmic membrane, activation by Na⁺ ions, and Western blot analysis with antiserum raised against the K. pneumoniae oxaloacetate decarboxylase. Participation of an oxaloacetate decarboxylase Na⁺ pump in l(+)-tartrate degradation by S. typhimurium is in accord with results from DNA analyses. The deduced protein sequence of the open reading frame identified upstream of the recently sequenced oxaloacetate decarboxylase genes is clearly homologous with the -subunit of l-tartrate dehydratase from Escherichia coli. Southern blot analysis with S. typhimurium chromosomal DNA indicated the presence of probably more than one gene for oxaloacetate decarboxylase.     

Molecular properties and sensitivity to cations of β-Galactosidase from Streptococcus thermophilus with four enzyme substrates

Summary -Galactosidase (E.C. 3.2.1.23) from an autolytic strain of Streptococcus thermophilus was purified to near homogeneity (466 U/mg protein). The quaternary structure of the -galactosidase was complex, the enzyme apparently existing in three forms in solution (as determined by HPLC ion-exchange or gel permeation chromatography). One form of the enzyme was stable but a second form dissociated with time in a temperature- and concentration-dependent manner to give the stable form. A single subunit was identified with a molecular weight of 116 000.Activation of enzyme activity by cations (Mg²⁺, K⁺ and Na⁺) was complex and varied markedly according to whether o-nitrophenyl--d-galactopyranoside (ONPG), d-nitrophenyl--d-galactopyranoside (PNPG), 4-methylumbelliferyl--d-galactopyranoside (4MeUmG) or lactose was the enzyme substrate. With all substrates there was synergistic activation with either Mg⁺⁺ and K⁺ or Mg⁺⁺ and Na⁺. Na⁺ was the better activator with either ONPG or 4MeUmG as the substrate while K⁺ was the better activator of lactose and PNPG hydrolysis. With either ONPG or 4MeUmG as substrate, and in the presence of both Mg²⁺ and K⁺, Na⁺ further enhanced activity. In contrast, Na⁺ was a competitive inhibitor of the Mg²⁺ and K⁺ activated reaction with either lactose or PNPG as the substrate. Analysis of the effect of the cations on the kinetics of lactose hydrolysis showed they all acted by increasing the binding of lactose of the enzyme as well as by increasing the maximum activity. Weak competitive inhibition of activity (with lactose) by galactose was found with a K_i of 350 mM galactose.     

Characterization of Na+ transport in normal human fibroblasts and neoplastic H.Ep.2 cells and the role of inhibitin

Summary Na⁺ transport was characterized in normal human fibroblasts and neoplastic H.Ep. 2 cells in order to investigate the role of the endogenous peptidic factor inhibitin that is secreted by a variety of neoplastic cells (including H.Ep. 2) and inhibits Na⁺/Na⁺ exchange in human erythrocytes. Although active (Na⁺, K⁺-ATPase mediated) Na⁺ fluxes were similar in the two cell types, H.Ep. 2 cells maintained higher intracellular Na[su+] concentration (26mm) compared to fibroblasts (12mm). An analysis of passive Na⁺ fluxes showed a difference in the handling of Na⁺ via ouabain and bumetanide-insensitive transport between the two cell types: H.Ep. 2 cells achieved net Na⁺ influx via an amiloride-sensitive pathway that was only demonstrated in fibroblasts when 10% fetal calf serum (FCS) was present. Kinetic studies were undertaken to investigate the interaction between Na⁺ flux via Na⁺/H⁺ and Na⁺/Na⁺ exchanges. for this purpose, an outwardly directed Na⁺ gradient was created by loading the cells with Na⁺ (Na _i >100mm) to activate the reverse functioning of Na⁺/H⁺ exchange (i.e., Na _out ⁺ H _in ⁺ ). The rates of ouabain-and bumetanide-insensitive Na⁺ efflux were measured over a range of extracellular Na⁺ concentrations (Na _o ⁺ 14–140mm). In the presence of 10% FCS, the two cell types showed different responses: in fibroblasts the Na⁺ efflux rate showed an inverse correlation with extracellular Na⁺ concentration, while H.Ep. 2 cells significantly increased their rate of Na⁺ efflux as extracellular Na⁺ concentration increased. So although the thermodynamic force would direct net Na⁺ efflux when Na _i ⁺ >Na _o ⁺ , H.Ep.2 cells were under kinetic control to perform Na⁺/Na⁺ exchange.When exogenous inhibitin was tested on fibroblasts, the steady-state intracellular Na⁺ concentration increased from 14 to 19mm (p<0.01). In Na⁺-loaded fibroblasts, serum-stimulated Na⁺ efflux was partially inhibitin sensitive and the maximal inhibitory effect was seen when extracellular Na⁺ concentration was 14mm and presumably the Na⁺/H⁺ exchanger operating in the reverse mode. This study demonstrated that, in contrast to fibroblasts, H.Ep.2 cells have a modified Na⁺/H⁺ exchange system whereby it acts in the Na _in ⁺ H _out ⁺ mode without exogenous growth factor activation and resists functioning in the reversed mode. It is proposed that inhibitin, is the endogenous modifier of this transport system in H.Ep.2 cells with the result that H.Ep.2 cells maintain a higher concentration of intracellular Na⁺ compared to fibroblasts.     

High-affinity sodium-dependent uptake of ascorbic acid by rat osteoblasts

Summary Ascorbic acid is essential for the formation of bone by osteoblasts, but the mechanism by which osteoblasts transport ascorbate has not been investigated previously. We examined the uptake ofl-[¹⁴C]ascorbate by a rat osteoblast-like cell line (ROS 17/2.8) and by primary cultures of rat calvaria cells. In both systems, cells accumulatedl-[¹⁴C]ascorbate during incubations of 1–30 min at 37°C. Unlike propionic acid, which diffuses across membranes in protonated form, ascorbic acid did not markedly alter cytosolic pH. Initial ascorbate uptake rate saturated with increasing substrate concentration, reflecting a high-affinity interaction that could be described by Michaelis-Menten kinetics (apparentK _m =30±2 m andV _max=1460±140 nmol ascorbate/g protein/min in ROS 17/2.8 cells incubated with 138mm extracellular Na⁺). Consistent with a stereoselective carrier-mediated mechanism, unlabeledl-ascorbate was a more potent inhibitor (IC₅₀=30±5 m) ofl-[¹⁴C]ascorbate transport than wasd-isoascorbate (IC₅₀=380±55 m). Uptake was dependent on both temperature and Na⁺, since it was inhibited by cooling to 4°C and by substitution of K⁺, Li⁺ or N-methyl-d-glucamine for extracellular Na⁺. Decreasing the external Na⁺ concentration lowered both the affinity of the transporter for ascorbate and the apparent maximum velocity of transport. We conclude that osteoblasts possess a stereoselective, high-affinity, Na⁺-dependent transport system for ascorbate. This system may play a role in the regulation of bone formation.     

Pump-leak sodium fluxes in low salt corn roots

Summary The influx and efflux of sodium from 4-hr washed, low salt corn roots (Zea mays L.) has been studied for characterization of passive and active components. Initial Na⁺ content of the roots is very low, 2.25±0.4 mol/g fresh weight. Na⁺ influx in the presence of 0.2mm Ca²⁺ and 0.002 to 20mm K⁺ is passive (a leak) based upon Goldman-type models, being determined by Na⁺ and cell potential (). Na⁺ was not transported by the K⁺ carrier and influx was unaffected by 50 m dicyclohexylcarbodiimide (DCCD). Permeability of the cells to Na⁺ was of the same order asP _k.Efflux of Na⁺ was by an efficient and rapid active transport system (a pump), thus accounting for the failure of these roots to accumulate high levels of Na⁺. In short-term loading and efflux experiments, internal Na⁺ turnover had a half-time of about 5 min. Sodium efflux was unaffected by DCCD. Net H⁺ flux was zero in the presence of DCCD regardless of sodium efflux, indicating absence of Na⁺/H⁺ antiport. Efflux of Na⁺ was equally rapid into medium containing no Na⁺ and only 0.002mm K⁺. K⁺ influx accounted for less than 4% of Na⁺ efflux, prompting the hypothesis that the Na⁺ (or cation?) efflux pump is the second electrogenic system previously defined based upon electrophysiological measurements.     

Function and presumed molecular structure of Na+-D-glucose cotransport systems

Functional characterization of Na⁺-d-glucose cotransport in intestine and kidney indicates the existence of heterogeneous Na⁺-d-glucose cotransport systems. Target size analysis of the transporting unit and model analysis of substrate binding have been performed and proteins have been cloned which mediate (SGLT1) and modulate (RS1) the expression of Na⁺-d-glucose cotransport. The experiments support the hypothesis that functional Na⁺-d-glucose cotransport systems in mammals are composed of two SGLT1-type subunits and may contain one or two RS1-type proteins. SGLT1 contains up to twelve membrane-spanning -helices, whereas RS1 is a hydrophilic extracellular protein which is anchored in the brush-border membrane by a hydrophobic -helix at the C-terminus. SGLT1 alone is able to translocate glucose together with sodium; however, RS1 increases the V _max of transport expressed by SGLT1. In addition, the biphasic glucose dependence of transport, which is typical for kidney and has been often observed in intestine, was only obtained after coexpression of SGLT1 and RS1.