Amino acid and22Na+ uptake in membrane vesicles from confluent simian virus 40 transformed Balb/c3T3 and Balb/c3T3

Summary The studies reported here were carried out to characterize further previously described changes in membrane localized amino acid transport associated with simian virus 40 transformation of the mammalian cell line, Balb/c3T3. Membrane vesicles were prepared from confluent cultures of both simian virus 40 transformed Balb/c3T3 (SV3T3) and the untransformed parent line, Balb/c3T3 (3T3). An initial, externally imposed out>in, 100mm Na⁺ gradient produces acceleration of early ingress of -aminoisobutyric acid (AIB) in vesicles from both cell lines, but transient, concentrative uptake (overshooting) only in SV3T3 vesicles. Early ingress ofl-leucine is also accelerated in SV3T3 vesicles by a Na⁺ gradient, and overshooting is also demonstrable.Na⁺-gradient independent AIB permeability of SV3T3 and 3T3 membranes was estimated using uptake data, a first order rate equation and measurements of vesicle size derived from quasi-elastic light-scattering studies. AIB permeability of SV3T3 membranes is greater than that of 3T3 membranes (113 Å/min and 43 Å/min, respectively), suggesting that overshooting in 3T3 vesicles is not attenuated by a Na⁺-independent AIB leak. Na⁺ permeability of the two membranes is similar, ruling out the possibility that a slower rate of Na⁺ equilibration across the SV3T3 membrane allows development of the overshoot.In SV3T3 vesicles the height of a Na⁺-gradient dependent overshoot varies with the initial [Na⁺] _o /[Na⁺] _i ratio, and [Na⁺] _o /[Na⁺] _i is linearly related to ln AIB uptake at overshoot peak/AIB uptake at equilibrium, consistent with the possibility that for [Na⁺] _o /[Na⁺] _i ratios in the range studied, AIB overshoot is energized by a constant proportion of the energy available from the initial electrochemical gradient for Na⁺.These results are consistent with the possibility that Na⁺-gradient dependent overshooting in SV3T3 vesicles is produced by Na⁺-amino acid carrier interactions resulting in either an increase in maximum transport velocity or an incrase in carrier affinity for AIB.Abbreviations used 3T3 Balb/c3T3 - SV3T3 simian virus 40 transformed Balb/c3T3 - AIB -aminoisobutyric acid     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (β-alanyl-l-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na⁺, K⁺ or choline⁺ gradient conditions (extravesicular > intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na⁺-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a K_m of 9.6 ± 1.4 mM and a V_max of 2.9 ± 0.2 nmol / mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-l-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na⁺-independent process.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium,Vibrio costicola

Active transport of -aminoisobutyric acid (AIB) in Vibrio costicola utilizes a system with affinity for glycine, alanine and, to some extent, methionine. AIB transport was more tolerant of high salt concentrations (3–4 M NaCl) in cells grown in the presence of 1.0 M NaCl than in those grown in the presence of 0.5 M NaCl. The former cells could also maintain much higher ATP contents than the latter in high salt concentrations.Transport kinetic studies performed with bacteria grown in 1.0 M NaCl revealed three effects of the Na⁺ ion: the first effect is to increase the apparent affinity (K _t) of the transport system for AIB at Na⁺ concentrations <0.2 M, the second to increase the maximum velocity (V _max) of transport (Na⁺ concentrations between 0.2 and 1.0 M), and the third to decrease the V _max without affectig K _t (Na⁺ concentrations >1.0 M). Cells grown in the presence of 0.5 M or 1.0 M NaCl had similar affinity for AIV. Thus, the differences in salt response of transport in these cells do not seem due to differences in AIB binding. Large, transport-inhibitory concentrations of NaCl resulted in efflux of AIB from cells preloaded in 0.5 M or 1.0 M NaCl, with most dramatic efflux occurring from the cells whose AIB transport was more salt-sensitive. Our results suggest that the degree to which high salt concentrations affect the transmembrane electrochemical energy source used for transport and ATP synthesis is an important determinant of salt tolerance.Abbreviations AIB -aminoisobutyric acid - pmf proton motive force     

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.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

Reconstitution and characterization of a sodium-stimulated active aminoisobutyric acid transport system derived from partially purified plasma membranes from mouse fibroblasts transformed by simian virus 40: comparison of reconstituted vesicles with native membrane vesicles

A Na⁺-specific and Na⁺-stimulated active α-aminoisobutyric acid transport system was reconstituted from plasma membranes isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40. The plasma membranes were treated with dimethylmaleic anhydride and then extracted with 2% cholate. The cholate-solubilized supernatant proteins were combined with exogenous phospholipids and eluted through a Sephadex G-50 column. This yielded reconstituted vesicles which in the presence of Na⁺ could actively transport α-aminoisobutyric acid as shown by the transient accumulation above the equilibrium level (overshoot). The overshoot was not obtained with other monovalent cations such as K⁺, Li⁺, and choline⁺. The electrochemical effect of the lipophilic anion, SCN^?, led to greater α-aminoisobutyric acid uptake as compared to that observed with Cl^? or SO₄^2?. The Na⁺-stimulated transport of a-aminoisobutyric acid was a saturable process with an apparent K_m of 2 mm. Studies of the inhibition of α-aminoisobutyric acid transport by other amino acids showed that methylaminoisobutyric acid [specifically transported by A system (alanine preferring)]had a pronounced inhibitory effect on a-aminoisobutyric acid uptake in contrast to the slight inhibitory effect produced by phenylalanine [primarily transported by L system (leucine preferring)]. The results show that the reconstituted vesicles, prepared from partially purified membrane proteins and exogenous phospholipids, regained the same important transport properties of native membrane vesicles, i.e., Na⁺-specific and Na⁺-stimulated concentrative α-aminoisobutyric acid uptake.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.     

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased bothK _m andV _max for GABA uptake, with an increase in theV _max/K _m ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 M GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 M ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 M veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na⁺, K⁺-ATPase acting as an ion pump.     

Na+/H+ antiport activity in tonoplast vesicles isolated from sunflower roots induced by NaCl stress

Na⁺ transport across the tonoplast and its accumulation in the vacuoles is of crucial importance for plant adaptation to salinity. Mild and severe salt stress increased both ATP- and PP_i-dependent H⁺ transport in tonoplast vesicles from sunflower seedling roots, suggesting the possibility that a Na⁺/H⁺ antiport system could be operating in such vesicles under salt conditions (E. Ballesteros et al. 1996. Physiol. Plant. 97: 259–268). During a mild salt stress, Na⁺ was mainly accumulated in the roots. Under a more severe salt treatment, Na⁺ was equally distributed in shoots and roots. In contrast to what was observed with Na⁺, all the salt treatments reduced the shoot K⁺ content. Dissipation by Na⁺ of the H⁺ gradient generated by the tonoplast H⁺-ATPase, monitored as fluorescence quenching of acridine orange, was used to measure Na⁺/H⁺ exchange across tonoplast-enriched vesicles isolated by sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots treated for 3 days with different NaCl regimes. Salt treatments induced a Na⁺/H⁺ exchange activity, which displayed saturation kinetics for Na⁺ added to the assay medium. This activity was partially inhibited by 125 μM amiloride, a competitive inhibitor of Na⁺/H⁺ antiports. No Na⁺/H⁺ exchange was detected in vesicles from control roots. The activity was specific for Na⁺. since K⁺ added to the assay medium slightly dissipated H⁺ gradients and displayed non-saturating kinetics for all salt treatments. Apparent K_m for Na⁺/H⁺ exchange in tonoplast vesicles from 150 mM NaCl-treated roots was lower than that of 75 mM NaCl-treated roots, V_max remaining unchanged. The results suggest that the existence of a specific Na⁺/H⁺ exchange activity in tonoplast-enriched vesicle fractions, induced by salt stress, could represent an adaptative response in sunflower plants, moderately tolerant to salinity.     

Transport of potassium inChara australis: II. Kinetics of a symport with sodium

Summary An electrogenic K⁺–Na⁺ symport with a high affinity for K⁺ has been found inChara (Smith & Walker, 1989). Under voltage-clamp conditions, the symport shows up as a change in membrane current upon adding either K⁺ or Na⁺ to the bathing medium in the presence of the other. Estimation of kinetic parameters for this transport has been difficult when using intact cells, since K⁺–Na⁺ current changes show a rapid falling off with time at K⁺ concentrations above 50 m. Cytoplasm-enriched cell fragments are used to overcome this difficulty since they do not show the rapid falling off of current change seen with intact cells. Current-voltage curves for the membrane in the absence or presence of either K⁺ or Na⁺ are obtained, yielding difference current-voltage curves which isolate the symport currents from other transport processes. The kinetic parameters describing this transport are found to be voltage dependent, withK _m for K⁺ ranging from 30 down to 2 m as membrane potential varies from –140 to –400 mV, andK _m for Na⁺ ranging between 470 and 700 m over a membrane potential range of –140 to –310 mV.Two different models for this transport system have been investigated. One of these involves the simultaneous transport of both the driver and substrate ions across the membrane, while the other allows for the possibility of the two ions being transported consecutively in two distinct reaction steps. The experimental results are shown to be consistent with either of these cotransport models, but they do suggest that binding of K⁺ occurs before that of Na⁺, and that movement of charge across the membrane (the voltage-dependent step) occurs when the transport protein has neither K⁺ nor Na⁺ bound to it.     

Electrogenic properties of the cloned Na+/glucose cotransporter: II. A transport model under nonrapid equilibrium conditions

Summary The results of the accompanying electrophysiological study of the cloned Na⁺/glucose cotransporter from small intestine (Parent, L., Supplisson, S., Loo, D.D.F., Wright, E.M. (1992) J. Membrane Biol. 125:49–62) were evaluated in terms of a kinetic model. The steady-state and presteady-state cotransporter properties are described by a 6-state ordered kinetic model (mirror symmetry) with a Na⁺:MDG stoichiometry of 2. Carrier translocation in the membrane as well as Na⁺ and sugar binding and dissociation are treated as a function of their individual rate constants. Empty carrier translocation and Na⁺ binding/ dissociation are the only steps considered to be voltage dependent. Currents were associated with the translocation of the negatively charged carrier in the membrane. Negative membrane potential facilitates sugar transport. One numerical solution was found for the 14 rate constants that account quantitatively for our experiment observations: i.e., (i) sigmoidal shape of the sugar-specific current-voltage curves (absence of outward currents and inward current saturation at high negative potentials), (ii) Na⁺ and voltage dependence of K _0.5 ^sugar and i _max ^sugar , (iii) sugar and voltage dependence of K _0.5 ^Na and i _max ^Na , (iv) presteady-state currents and their dependence on external Na⁺, MDG and membrane potential, and (v) and carrier Na⁺ leak current. We conclude that the main voltage effect is on carrier translocation. Na⁺ ions that migrate from the extracellular medium to their binding sites sense 25 to 35% of the transmembrane voltage, whereas charges associated with the carrier translocation experiences 60 to 75% of the membrane electrical field. Internal Na⁺ ion binding is not voltage dependent. In our nonrapid equilibrium model, the rate-limiting step for sugar transport is a function of the membrane potential, [Na]₀ and [MDG]₀. At 0 mV and at saturating [Na]₀ and [MDG]₀, the rate-limiting step for sugar transport is the empty carrier translocation (5 sec^–1). As the membrane potential is made more negative, the empty carrier translocation gets faster and the internal Na⁺ dissociation becomes increasingly rate limiting. However, as [Na]₀ is decreased to less than 10 mm, the rate-limiting step is the external Na⁺ ions binding in the 0 to –150 mV potential range. At 0 mV, the external Na⁺ dissociation constant KNa is 80 mm and decreases to 24 mm at –150 mV. The external sugar dissociation constant KNaS is estimated to be 200 m and voltage independent. Finally, the internal leak pathway (CNa₂ translocation) is insignificant. While we cannot rule out a more complex kinetic model, the electrical properties of the cloned Na⁺/glucose cotransporter are found to be adequately described by this 6-state kinetic model.We are grateful to Drs. A. Berteloot, S. Ciani, and J.-Y. Lapointe for stimulating discussions and thank our colleagues for comments. L.P. was recipient of a post-doctoral fellowship from the Medical Research Council of Canada. This work was supported by a grant from the U.S. Public Health Service DK 19567.     

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

Summary Time courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na⁺ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na⁺) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na⁺) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na⁺ on both membrane sides and with a clamped membrane potential,K _D values of 0.4 and 7.9 M were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence ofd-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na⁺(out>in). Low and high affinity transport could be fitted with identicalK _m values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparentK _m ofhigh affinity transport whereas the apparentK _m of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pighigh and low affinity Na⁺-d-glucose cotransporters are present which containlow and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport moleculc.     

Phosphate transport in intestinal brush-border membrane

In the small intestine of the rabbit the process of Na⁺-dependent uptake of phosphate occurs only at the brush-border of duodenal enterocytes. Li⁺ can replace Na⁺. The process is activated when either K⁺, Cs⁺, Rb⁺, or choline is present in the intravesicular space. The presence of membrane-permeable anions is essential for maximum rates of phosphate transport. We conclude that the mechanism of the phosphate carrier is electrogenic at pH 6–8, probably two Na⁺ moving with each H₂PO ₄ ^– . This. will lead to the development of a positive charge within the vesicle. The variation of theK _m for H₂PO ₄ ^– with pH is thought to be the consequence of the affinity of the carrier protein for H₂PO ₄ ^– increasing as the pH increases. Polyclonal antibodies against membrane vesicles isolated from rabbit duodenum, jejunum, and ileum were prepared. The antibodies raised against the ileum and jejunum both activated the phosphate transport process, while the anti-duodenum antibody preparation inhibited phosphate transport.     

Lanthanide/proline cotransport across rabbit renal brush borders

Summary It has been suggested previously that La³⁺ can replace Na⁺ on various cotransport systems in renal brush border membranes. In the present study, we used rabbit renal brush border membrane vesicles to examine the specificity and kinetics of Ln³⁺/proline cotransport. Experiments were carried out under zero-trans, voltage clamped conditions using a rapid-mix/filtration technique. Initial experiments confirmed that La³⁺ produced the classical overshoot phenomenon. The initial rates of proline uptake relative to Na⁺ were Eu³⁺, Tb³⁺, Nd³⁺, Pr³⁺, Ho³⁺ (3.3)>Na⁺ (1.0)>La³⁺ (0.86) > choline⁺ (0.1). At a saturating salt concentration, uptake saturated with increasing proline concentration: theK _t andJ _max were 0.05mm and 17 pmol mg^–1 sec^–1 in Na⁺; and 0.28mm and 73 pmol mg^–1 sec^–1 in Tb³⁺. The higherJ _max in Tb³⁺ indicates that the Tb³⁺-proline loaded carrier is more effective than the Na⁺-proline loaded carrier in overcoming some rate-limiting barriers in the transport process. Na⁺ activated proline uptake with a Hill coefficient of 1.6 and aK _0.5 of 21mm, while Tb³⁺ activated with a Hill coefficient of 0.88 and aK _0.5 of 28mm. The Hill coefficient for Na⁺ suggests two binding sites, whereas the Hill coefficient for Tb³⁺ may indicate negative cooperativity between the trivalent ligands at the binding sites. We conclude that lanthanides are able to substitute for Na⁺ on the brush border proline carrier and that the lanthanides may serve as useful probes for the ligand binding sites.     

Phenylalanine uptake in isolated renal brush border vesicles

The uptake of l-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques.Brush border microvilli but not basolateral plasma membrane vesicles take up l-phenylalanine by an Na⁺-dependent, saturable transport system. The apparent affinity of the transport system for l-phenylalanine is 6.1 mM at 100 mM Na⁺ and for Na⁺ 13 mM at 1 mM l-phenylalanine. Reduction of the Na⁺ concentration reduces the apparent affinity of the transport system for l-phenylalanine but does not alter the maximum velocity.In the presence of an electrochemical potential difference for Na⁺ across the membrane (η_Na0 >η_Na1) the brush border microvilli accumulate transiently l-phenylalanine over the concentration in the incubation medium (overshoot phenomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K⁺ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient.These results indicate that the entry of l-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na⁺ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na⁺ and on the electrical potential difference. The exit of l-phenylalanine across the basolateral plasma membranes is Na⁺-independent and probably involves facilitated diffusion.     

Valine uptake by the scleractinian coral Galaxea fascicularis: characterization and effect of light and nutritional status

The characteristics of valine uptake by isolated microcolonies of Galaxea fascicularis (Linnaeus 1758) were studied under various conditions including light, dark and feeding. The results demonstrated the presence of: (1) a linear component which might represent either a diffusional transport or a low-affinity carrier-mediated transport (apparent carrier affinity >250 mol·l^–1), and (2) a high-affinity active carrier-mediated transport (apparent carrier affinity about 5 mol·l^-1). The latter is mediated by two different systems: (i) a Na⁺-dependent carrier, stimulated by light and operative in both fed and unfed polyps, and (ii) a Na⁺-independent carrier, light insensitive and present only in unfed polyps. Competition experiments with other amino acids show that the Na⁺-dependent carrier is highly specific for neutral amino acids, as indicated by the high inhibition constants of basic and acidic amino acids. Our results suggest that the energy supplied by zooxanthellae photosynthates is necessary for the process of amino acid uptake, and that the Na⁺-dependent carrier responsible for valine uptake by G. fascicularis is similar to the B^0,+ system.Abbreviations AA amino acid(s) - AC/HC ratio autotrophic/heterotrophic carbon - ASW artificial sea water - DOM dissolved organic material - HPLC high performance liquid chromatography - K ₁ apparent inhibition constant - K _m apparent affinity of the carrier - SE standard error - V _max maximal rate of absorption