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1.
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.  相似文献   

2.
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.  相似文献   

3.
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.  相似文献   

4.
Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK m of 0.12±0.02mm andV max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na+-dependent, the cation not being replaceable with Li+, K+, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl appeared to be necessary for full activation of Na+-dependent glutamine transport. Two external Na+ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na+/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na+-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B0,+.  相似文献   

5.
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.  相似文献   

6.
It is proposed that the activity of an epidermal cotransport system for Na+ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na+-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na+-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K m for L-glutamate uptake was 146 mol·l-1, and the maximum velocity of uptake (V max) was 12.1 pmol·mm-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l-1 and 8.4 pmol·mm-2·min-1. The Na+/L-glutamate co-transporter has a stoichiometry of at least two Na+ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na+ equivalent to a K m of 21 mmol · l-1 Na+. Na+ is the only external ion apparently required to drive L-glutamate uptake. Li+ substitutes weakly for Na+. Removal of external K+ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na+/K+ gradient by inhibiting epidermal Na+/K+ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na+-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na+]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K m Michaelis constant - n app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V max maximum velocity of uptake  相似文献   

7.
Summary The substrate stoichiometry of the intestinal Na+/phosphate cotransporter was examined using two measures of Na+-dependent phosphate uptake: initial rates of uptake with [32P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC3(5). Isotopic phosphate measures electrogenic and electroneutral Na+-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na+/phosphate cotransport was found to have similar Na+ activations (apparentK 0.5's of 28 and 25mm), apparentK m 's for phosphate (100 and 410 m), andK 0.5's for inhibition by phenylglyoxal (70 and 90 m) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na+-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na+ with apparentK 0.5 for phosphate of 200 m; similar to the apparentK m for phosphate uptake, andK 0.5 for phosphate protection against FITC-PG inhibition of Na+-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na+/phosphate cotransporter is electrogenic at pH 7.4, that H2PO 4 is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na+/phosphate cotransporter.  相似文献   

8.
Summary Ehrlich ascites tumor cells contain a Na+ uptake system, which is activated by internal protons and is inhibited by amiloride with an IC50 of 25 m and by dimethylamiloride with an IC50 of 0.6 m at 1mm external Na+. Decrease of external Na+ or addition of amiloride is followed by a decrease of internal pH. Taken together, these findings suggest the presence of an operative Na+/H+ antiport system, which is involved in the regulation of internal pH. We cannot find a significant contribution of a proton pump activated by glycolysis to the pH gradient. At an external pH between 7.0 and 7.6, quiescent cells are more alkaline than exponentially growing cells (0.1 to 0.17 units). Accordingly, an increase of the affinity of the Na+/H+ antiport for internal protons in quiescent cells is demonstrated by the following findings: 1. The internal pH, at which the half-maximal activation of the amiloride-sensitive Na+ uptake occurs, is shifted from 6.85 to 7.1 at 1mm external Na+. 2. The threshold value of external pH, below which a pronounced effect of amiloride on steadystate internal pH is observed, is shifted from 7.0 in growing to 7.5 in quiescent cells at physiological Na+ concentrations. Therefore, we conclude that quiescent Ehrlich ascites tumor cells raise their internal pH by increasing the affinity of their Na+/H+ antiporter to internal protons. The Na+/H+ antiport cannot be activated further by addition of serum growth factors to quiescent cells. All experiments were performed at bicarbonate concentrations in the medium which do not exceed 0.5mm. The data are discussed in view of existing models of mitogenic activity of transitory pH changes.  相似文献   

9.
Summary Na, K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na+ transport during early postnatal development. The maximum Na+-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (I sc N ) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats.I sc N was entirely inhibited by ouabain; the inhibitory constant was 0.1mm in 10-day-old (young) and 0.4mm in 90-day-old (adult) rats. The affinity of the Na, K pump for Na+ was higher in young (11mm) than in adult animals (19mm). The Na, K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (K I ) was not changed during development (0.1–0.3mm). Specific [3H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (K D ) was 8 and 21 m in young and adult rats, respectively. The Na+ turnover rate per single Na, K pump, which was calculated fromI sc N and estimated density of binding sites per cm2 of tissue was 500 in adult and 6400 Na+/min·site in young rats. These data indicate that the very high Na+ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na+ of a single isoform of the Na, K pump. The development of Na+ extrusion across the basolateral membrane is not directly regulated by corticosteroids.  相似文献   

10.
Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 cm2. The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm Na+, and serosal Na+ is substituted by K+, Cs+, or N-methyl-d-glucamine, the basolateral membrane expresses what appears to be a large Na+ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca2+. In Cl-free SO 4 2– Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K+. This pathway can be blocked by serosal amiloride (K i=13.1 m) or serosal Na+ ions (K i 10 to 20mm). It also conducts Li+ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec–1 at 0 mV.  相似文献   

11.
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 B0,+ 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  相似文献   

12.
Nitrosomonas europaea is capable of incorporating exogenously supplied amino acids. Studies in whole cells revealed that at least eight amino acids are actively accumulated, probably by the action of three different transport systems, each with high affinity ( molar range) for several amino acids. Evidence for the action of secondary mechanisms of transport was obtained from efflux, counterflow and exchange experiments. More detailed information was obtained from studies in liposomes in which solubilized integral membrane proteins of N. europaea were incorporated. Uptake of l-alanine in these liposomes could be driven by artificially imposed pH gradients and electrical potentials, but not by chemical sodium-ion gradients. These observations indicate that l-alanine is transported by a H+/alanine symport system. The ecological significance of secondary amino acid transport systems in autotrophic ammonium-oxidizing bacteria is discussed.  相似文献   

13.
Summary We previously reported that3H-folate uptake by rabbit jejunal brush-border membrane (BBM) vesicles was markedly stimulated by an outwardly directed OH gradient (pHin 7.7, pHout 5.5), inhibited by anion exchange inhibitors (DIDS, SITS, furosemide), and saturable (folateK m=0.19 m) suggesting carrier-mediated folate/OH exchange (or H+/folate cotransport). In the present study, the anion specificity of this transport process was examined. Under conditions of an outwardly directed OH gradient, DIDS-sensitive folate uptake wascis inhibited (>90%) by reduced folate analogues: dihydrofolate (IC50=0.40 m), folinic acid (IC50=0.50 m), 5-methyltetrahydrofolate (IC50=0.53 m), and (+)amethopterin (IC50=0.93 M). In contrast, 10 m (–)amethopterin had only a modest effect on folate uptake (18% inhibition) suggesting stereospecificity of the folate/OH exchanger. The nonpteridine compounds which are transported by the folate carrier in L1210 leukemic cells (phthalate, thiamine pyrophosphate, and PO 4 –3 ) did not inhibit jejunal folate uptake. Furthermore, folate uptake was not inhibited by SO 4 –2 (4mm) or oxalate (4mm) thereby distinguishing this carrier from the previously described intestinal SO 4 –2 /OH and oxalate/Cl exchangers. After BBM vesicles were loaded with3H-folate, the initial velocity of3H-folate efflux was stimulated by unlabeled folate in the efflux medium. The transstimulation of3H-folate efflux by unlabeled folate was furosemide (or DIDS) inhibitable and temperature sensitive. Half-maximal stimulation of furosemide-sensitive3H-folate efflux was observed with 0.25±0.05 m unlabeled folate, a concentration similar to theK m for folate uptake. These data suggest that folate-stimulated3H-folate efflux is mediated by the folate/OH exchanger. With the exception of (–) amethopterin, reduced folate analogues also transstimulated furosemide-sensitive3H-folate efflux in a concentration-dependent manner suggesting stereospecific transport of these analogues by the folate/OH exchanger. In summary, folate transport by the jejunal folate/OH exchanger demonstrates bothcis inhibition and transstimulation by reduced folate analogues, but not by other inorganic or organic anions suggesting bidirectional transport of folate and a high degree of anion specificity.  相似文献   

14.
Summary A simple procedure was developed for the isolation of a sarcolemma-enriched membrane preparation from homogenates of bullfrog (Rana catesbeiana) heart. Crude microsomes obtained by differential centrifugation were fractionated in Hypaque density gradients. The fraction enriched in surface membrane markers consisted of 87% tightly sealed vesicles. The uptake of86Rb+ by the preparation was measured in the presence of an opposing K+ gradient using a rapid ion exchange technique. At low extravesicular Rb+ concentrations, at least 50% of the uptake was blocked by addition of 1mm ouabain to the assay medium. Orthovanadate (50 m), ADP (2.5mm), or Mg (1mm) were also partial inhibitors of Rb+ uptake under these conditions, and produced a complete block of Rb+ influx in the presence of 1mm ouabain. When86Rb+ was used as a tracer of extravesicular K+ (Rb 0 + 40 m K 0 + =0.1–5mm) a distinct uptake pathway emerged, as detected by its inhibition by 1mm Ba2+ (K 0.5=20 m). At a constant internal K+ concentration (K in + =50mm) the magnitude of the Ba2+-sensitive K+ uptake was found to depend on K 0 + in a manner that closely resembles the K+ concentration dependence of the background K+ conductance (I Kl) observed electrophysiologically in intact cardiac cells. We conclude that K+ permeates passively this preparation through two distinct pathways, the sodium pump and a system identifiable as the background potassium channel.  相似文献   

15.
Summary The amino acid pool of MDCK cells was essentially constituted by alanine, glycine, glutamic acid, serine, taurine, lysine, -alanine and glutamine. Upon reductions in osmolarity, free amino acids were rapidly mobilized. In 50% hyposmotic solutions, the intracellular content of free amino acids decreased from 69 to 25mm. Glutamic acid, taurine and -alanine were the most sensitive to hyposmolarity, followed by glycine, alanine and serine, whereas isoleucine, phenylalanine and valine were only weakly reactive. The properties of this osmolarity-sensitive release of amino acids were examined using3H-taurine. Decreasing osmolarity to 85, 75 or 50% increased taurine efflux from 0.6% per min to 1.6, 3.5 and 5.06 per min, respectively. The time course of3H-taurine release closely follows that of the regulatory volume decrease in MDCK cells. Taurine release was unaffected by removal of Na+, Cl or Ca2+, or by treating cells with colchicine or cytochalasin. It was temperature dependent and decreased at low pH. Taurine release was unaffected by bumetanide (an inhibitor of the Na+/K+/2Cl carrier); it was inhibited 16 and 67 by TEA and quinidine (inhibitors of K+ conductances), unaffected by gadolinium or diphenylamine-2-carboxylate (inhibitors of Cl channels) and inhibited 50% by DIDS. The inhibitory effects of DIDS and quinidine were additive. Quinidine but not DIDS inhibited taurine uptake by MDCK cells.  相似文献   

16.
Summary Ion dependence and electrogenicity of taurine uptake were studied in rabbit renal outer cortical brush-border membrane vesicles isolated by differential precipitation. Na+-d-glucose cotransport was followed in parallel to monitor changes in the membrane potential. Concentrative taurine flux was dependent on a chemical and/or an electrical Na+ gradient (K+ diffusion potential) and could be completely inhibited by other -amino acids. It displayed a specific anion requirement (ClBrSCN>I>NO 3 ). At chemical Na+ equilibrium, Cl gradients, depending on their orientation, stimulated or inhibited taurine uptake more than could be attributed solely to electrical anion effects, although a Cl gradient alone could not energize an overshoot. Furthermore, taurine tracer exchange was significantly stimulated by Cl as well as Br. The Cl stoichiometry was found to be one, whereas taurine transport, in the presence of Cl, was sigmoidally related to the Na+ concentration, resulting in a coupling ratio of 2 to 3 Na+: 1 taurine. Upon Cl replacement with gluconate, taurine uptake showed a reduced potential sensitivity and was no longer detectably affected by the Na+ concentration (up to 150mm). These results suggest a 2 to 3 Na+:1 Cl:1 taurine cotransport mechanism driven mainly by the Na+ gradient, which is sensitive to the membrane potential due to a negatively charged empty carrier. Cl appears to stimulate taurine flux primarily by facilitating the formation of the translocated solute-carrier complex.  相似文献   

17.
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.  相似文献   

18.
Summary Ion flux relations in the unicellular marine algaAcetabularia have been investigated by uptake and washout kinetics of radioactive tracers (22Na+,42K+,36Cl and86Rb+) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K+ with influx and efflux of about 100 nmol·m–2·sec–1×K+ passes three- to sevenfold more easily than Rb+ does. Under normal conditions, Cl (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mol·m–2·sec–1, and a cytoplasmic as well as vacuolar [Cl] of about 420mm ([Cl] o =529mm). At 4°C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl], are significantly reduced. Na+ ([Na+] i : about 70mm, [Na+] o : 461mm), which is of minor electrophysiological relevance compared to K+, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mol·m–2·sec–1 for influx and efflux). Some results with Na+ and Cl are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normalAcetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the central vacuole, with the cytoplasm and with the external medium.  相似文献   

19.
About 92% of the taurine influx in flounder erythrocytes at physiological conditions in vitro (330 mosmol·l-1, 145 mmol·l-1 Na+, 0.30 mmol·l-1 taurine) is Na+-dependent. This influx is highly specific for taurine. The -amino compounds hypotaurine and -alanine were the only compounds which mimicked the inhibitory effect of taurine on influx of [14C]taurine, the former more than the latter. Counterexchange of taurine was also mediated by the taurine transporters. Reduction of osmolality per se did not affect the activity of these transporters. Non-linear regression analysis of the influx values revealed the presence of two different influx systems: a system with high affinity and low capacity and another with low affinity and high capacity. However, we cannot exclude the possibility that this influx of taurine was mediated by only one transporter which operated in different modes depending on the extracellular Na+ concentration. On the assumption that the Na+-dependent influx was mediated by two separate systems, the maximal velocity of the low capacity system was 2.55 nmol·g dry weight-1·min-1 at 145 mmol·ll-1 extracellular Na+. This capacity was about 50% lower than that of the high capacity system. The Michaelis constants were 0.013 and 1.34 mmol·l-1, respectively. Reduction of the extracellular Na+ concentration reduced maximal velocity and the affinity to taurine of both transport systems. At 10 mmol·l-1 Na+ or lower concentrations the high capacity system did not seem to operate. The activation method suggested that each taurine molecule transported by the high capacity system was accompanied by two Na+. The stoichiometry of the low capacity system was 1 taurine: 1 Na+. The Hill-coefficient for both transport systems was 1.00.Abbreviations cpm counts per minute - dw dry weight - GABA -amino-n-butyric acid - K m Michaelis constant - pK b basic dissociation constant - SD standard deviation - -ABA Dl--amino-n-butyric acid - V max maximal velocity - ww wet weight  相似文献   

20.
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.  相似文献   

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