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1.
Summary We have studied current (I Str) through the Na, K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically,I Str was measured as the strophanthidin-sensitive current in the presence of Ba2–, Cd2+ and gluconate (in place of external Cl). In addition,I Str was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a meanK 0.5 of 0.4 m, with 2–5 m producing 84–93% pump' block. From baseline data with 12 Na+-preloaded oocytes, voltage clamped in the range [–170, +50 mV] with and without 2–5 m Str, the averageI Str depended directly onV m up to a plateau at 0 mV with interpolated zero current at –165 mV. In three oocytes, lowering the external [Na+] markedly decreased the voltage sensitivity ofI p , while producing only a small change in the maximal outwardI Str. In contrast, decreasing the external [K+] from 25 to 2.5mm reducedI Str at 0 mV without substantially affecting its voltage dependence. At K+ concentrations of 1mm, both the absolute value ofI Str at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averagedI Str by [K+] o over the voltage interval [–30, +30 mV] was well fit by the Hill equation, with K=1.7±0.4mm andnH (the minimum number of K+ binding sites) =1.7±0.4. The results unequivocally establish that the cardiotonic-sensitive current ofRana oocytes displays only a positive slope conductance for [K+] o >1mm. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na+ and K+ are consistent with results obtained in other tissues and may reflect an ion-well effect.  相似文献   

2.
Summary Addition of 0.1–0.3 m A23187, a divalent cation ionophore, to human erythrocytes suspended in a 1.0mm 45Ca2+-containing buffer results in a small ( two fold) increase in [Ca2+] i , a significant decrease in osmotic fragility, and a decrease in intracellular K+ (100 mmoles/liter of cells to 70 mmoles/liter cells) without significant alteration of intracellular [Na+]. This decrease in [K+] i is associated with a significant decrease in packed cell volume and correlates directly with the observed alteration is osmotic fragility. Increasing extracellular K+ to 125mm prevents the A23187-induced changes in osmotic fragility, K+ content and cell volume, but does not prevent the ionophore-induced uptake of45Ca2+. Addition of 0.1–0.3 m A23187 to toad erythrocytes leads to an increase in45Ca2+ uptake comparable to that observed in human erythrocytes, but does not alter osmotic fragility, cell volume or K+ content. Higher concentrations of ionophore (3.0–10.0 m) cause a 30- to 50-fold increase in45Ca2+ uptake and concomitant change in K+ content, cell volume and osmotic fragility. These changes in cell properties can be prevented by increasing extracellular [K+] to 90mm. The difference in sensitivity of the two cell types to A23187 is attributed to the presence of additional intracellular calcium pools within toad erythrocytes that prevent an increase in cytoplasmic Ca2+ until Ca2+ uptake is increased substantially at the higher concentrations of A23187.  相似文献   

3.
Summary We have confirmed previous demonstrations of sodium gradient-stimulated transport ofl-alanine, phenylalanine, proline, and -alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these14C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability ofd-glucose to inhibit tracer uptake.l-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the14C-l-alanine uptake could be inhibited by proline and -alanine, while lysine and MeAIB were no more effective thand-glucose. Roughly 50% of the14C-phenylalanine uptake could be inhibited by proline and -alanine; lysine was as effective as proline and -alanine, and the effects of pairs of these amino acids at 50mm each were not cumulative. MeAIB was no more effective thand-glucose. We conclude that three pathways mediate the uptake of neutral,l, -amino acids. One system is inaccessible to lysine, proline, and -alanine. The second system carries a major fraction of thel-alanine flux; it is sensitive to proline and -alanine, but not to lysine. The third system carries half the14C-phenylalanine flux, and it is sensitive to proline, lysine, and -alanine. Since the neutral,l, -amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classicalA system, and since all of thel-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classicalASC system.l-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective thand-glucose in inhibiting lysine uptake, while proline and -alanine appear to inhibit a component of the lysine flux. We conclude that the14C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, -alanine, andl-alanine, and one which is inhibited byl-alanine and phenylalanine but inaccessible to proline, -alanine, and MeAIB. Fluxes of14C-proline and14C-MeAIB are completely inhibited byl-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit14C--alanine uptake. However, -alanine inhibits only 38% of the14C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports -alanine.  相似文献   

4.
Summary The interaction of 1-anilino-8-naphthalene-sulfonate (ANS) with vesicles derived from hog fundic mucosa was studied in the presence of valinomycin and with the addition of ATP. Evidence was found for two classes of sites, those rapidly accessible to ANS with aK D of 7.5 m and those slowly accessible, but rapidly accessed in the presence of valinomycin with aK D of 2.5 m. ATP transiently increases the quantum yield of the latter ANS binding sites only in the presence of valinomycin, but does not alter the number ofK D of those sites. The time course of this increase correlates with H+ uptake and Rb+ extrusion by those vesicles and H+ carriers such as tetrachlorsalicylanilide or nigericin abolish the ATP response. With ATP addition in the presence of SC14N and valinomycin there is transient uptake of SCN. It is concluded that ANS is acting as a probe of a structural change dependent on a potential and H+ gradient.  相似文献   

5.
Summary In jejunal brush-border membrane vesicles, an outwardly directed OH gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M. 1985.J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH exchange (or phenomenologically indistinguishable H+: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examining F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was greater at lower (pHint/pHext: 5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive3H-folate uptake only at pH6.0. Since stepwise increments ininternal pH (4.57.5; pHext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH (4.356.5; pHint=7.8), a 50-fold decrement in F uptake was observed (H+ K m =12.8±1.2 M). Hill plots of these data suggest involvement of at least one H+ (OH) at low pH (monovalent F predominates) and at least 2 H+ (OH) at high pH (divalent F–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pHext 4.55 or 5.8, transport of F and F–2 is electroneutral. Kinetic parameters for F and F–2 were calculated from uptake data at pHext 4.55 and 5.0. Comparison of predictedvs. experimentally determined kinetic parameters at pHext5.8 (K m =1.33vs. 1.70 M;V max=123.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV max, but does not affect theK m for carrier-mediated F transport. These data are consistent with similarK i ' s for sulfasalazine (competitive inhibitor) at pHext 5.35 and 5.8 (64.7 and 58.5 M, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalent F and is sensitive to external pH with a H+ K m (or OH lC50) corresponding to pH 4.89. External pH effects theV max, but not theK m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH or H+),rather than competitive binding that is mutually exclusive.  相似文献   

6.
Summary Efflux of42K+ was measured in frog sartorius muscles equilibrated in depolarizing solutions with external K+ concentrations ([K+] o ) between 75 and 300mm and NaCl concentrations of 60, 120, or 240mm. For several combinations of KCl and NaCl, steady-state internal potentials (V i) were the same for different [K+] o . For the range ofV i examined, K+ efflux occurs principally through the K+ inward rectifier channels. When external K+ is removedV i remains constant for 2 to 3 hr because of the high membrane conductance to Cl, but K+ efflux drops by about one order of magnitude.External Ba2+ in the presence or absence of external K+ produces an inhibition of K+ efflux described by a relation of the formu=(u1/(1+C)[Ba2+] o ))+u 2, whereu is the uninhibited fraction of K+ efflux;u 1, u2 andC are constants; andu 1+u2=1.C depends both on [K+] o andV i. When [K+] o 75mm, increasing [K+] o at constantV i reduces Ba2+ sensitivity. For constantV i–30 mV, Ba2+ sensitivity is less when [K+] o =0 than when [K+] o 75mm. When [K+] o =0, Ba2+ sensitivity decreases asV i is made more positive. The dependence of the Ba2+ sensitivity onV i at constant [K+] o is greater when [K+] o =0 than when [K+] o 75mm.Both the activation of K+ efflux by external K+ and the Ba2+ inhibition of K+ efflux can be explained on the basis of two membrane control sites associated with each channel. When both sites are occupied by K+, the channels are in a high flux state. When one or both sites are empty, the channels are in a low, nonzero flux state. When Ba2+ occupies either site, K+ efflux is further reduced. The reduction of Ba2+-sensitivity by increasing [K+] o at high [K+] o is attributable to the displacement of Ba2+ from the control sites by K+. The increased Ba2+ sensitivity produced by going from [K+] o =0 to [K+] o >-75mm whenV i–30 mV is attributable to states in which Ba2+ occupies one site and K+ the other when [K+] o 0. The smallerV i dependence of the Ba2+ sensitivity when [K+] o 75mm compared to [K+] o =0 is attributable to the necessity that Ba2+ displace K+ at the control sites when [K+] o is high but not when [K+] o =0.  相似文献   

7.
The sequence of subfamilies,Cinchonoideae, Antirheoideae andRubioideae, attemps to show their natural affinities and phylogeny. The subfamilies are those ofVerdcourt, and the order in which they are presented is that ofBremekamp. A list is presented of the subfamilies, tribes and genera of theRubiaceae to be utilized in the Catálogo Ilustrado de las plantas de Cundinamarca, Colombia.  相似文献   

8.
In the light, 2 out of the 4 newly testedChlorella vulgaris strains were found to use about as many amino acids as a source of nitrogen as the previously investigated strain Delft; the other 2C. vulgaris strains and 3 of theAnkistrodesmus strains used only a few. The 4thAnkistrodesmus strain used none.On the average,l-amino acids supported better growth thand-amino acids, butd-serine was preferred tol-serine by 3Ankistrodesmus strains.In the dark, growth was only obtained withC. vulgaris strain Delft, and only on a few of thel-amino acids,l-leucine in particular.The author is indebted to the Direction of the Academic Hospital Dijkzigt, Rotterdam and to Prof. Dr. H. Esseveld, Head of the Central Bacteriological Laboratory, Rotterdam, for providing facilities for the performance of this study.He thanks Mrs. Dr. H. J. Leijnse-Ybema for her help in making the chromatograms, and Mr. J. B. Lenstra, pharmacist, for advice in matters of organic chemistry.  相似文献   

9.
Summary Micromolar concentrations of silver ion activate large Ca2+ fluxes across the plasma membrane of intact rod outer segments isolated from bovine retinas (intact ROS). The rate of Ag+-induced Ca2+ efflux from intact ROS depended on the Ag+ concentration in a sigmoidal manner suggesting a cooperative mechanism with a Hill coefficient between 2 and 3. At a concentration of 50 m Ag+ the rate of Ca2+ efflux was 7×106 Ca2+/outer segment/sec; this represents a change in total intracellular Ca2+ by 0.7mm/outer segment/sec. Addition of the nonselective ionophore gramicidin in the absence of external alkali cations greatly reduced the Ag+-induced Ca2+ efflux from intact ROS, apparently by enabling internal alkali cations to leak out. Adding back alkali cations to the external medium restored Ag+-induced Ca2+ efflux when gramicidin was present. In the presence of gramicidin, Ag+-induced Ca2+ efflux from intact ROS was blocked by 50 m tetracaine orl-cis diltiazem, whereas without gramicidin both blockers were ineffective. Bothl-cis diltiazem and tetracaine are blockers of one kinetic component of cGMP-induced Ca2+ flux across ROS disk membranes. The ion selectivity of the Ag+-induced pathway proved to be broad with little discrimination between the alkali cations Li+, Na+, K+, and Cs+ or between Ca2+ and Mg2+. The properties of the Ag+-induced pathway(s) suggest that it may reflect the cGMP-dependent conductance opened in the absence of cGMP by silver ions.  相似文献   

10.
Summary The properties of Ca2+-activated K+ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K+ over Na+ and K+ over Cs+. They were blocked by high cytoplasmic Na+ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca2+ concentrations. Increasing Ca2+ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca2+ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba2+ reduced both channel-open probability and conductance. The sites for the action of Ba2+ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba2+ dissociation constants (K d (0 mV) for the effects on probability and conductance, respectively. TEA+ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK d (0 mV)=5.6mm. A second site, with a higher affinity,K d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA+ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca2+-activated K+ channels found in other tissues.  相似文献   

11.
Summary Brush border membrane vesicles (BBMV) purified from steer jejunum were used to study the kinetics of sodiumd-glucose cotransport under voltage clamped, zero-trans conditions. When the initial rate of glucose transport (J gluc) was measured over a wide range of glucose concentrations ([S]=0.01–20mm), curvature of the Woolf-Augustinsson-Hofstee plots was seen, compatible with a diffusional and one major, high capacity (maximal transport rateJ max=5.8–8.8 nmol/mg·min) saturable system. Further studies indicated that changes incis [Na] altered theK t , but not theJ max, suggesting the presence of a rapid-equilibrium, ordered bireactant system with sodium adding first.Trans sodium inhibitedJ gluc hyperbolically. KCl-valinomycin diffusion potentials, inner membrane face positive, loweredJ gluc, while potentials of the opposite polarity raiseJ gluc. At low glucose concentrations ([S]<0.05mm), a second, minor, high affinity transport system was indicated. Further evidence for this second saturable system was provided by sodium activation curves, which were hyperbolic when [S]=0.5mm, but were sigmoidal when [S]=.0.01mm. Simultaneous fluxes of22Na and [3H]glucose at 1mm glucose and 30mm NaCl yielded a cotransport-dependent flux ratio of 21 sodium/glucose, suggestive of 11 (Na/glucose) high capacity, low affinity system and a 31 (Na/glucose) high affinity, low capacity system. Kinetic experiments with rabbit jejunal brush borders revealed two major Na-dependent saturable systems. Extravesicular (cis) Na changed theK t , but not theJ max of the major system.  相似文献   

12.
Murine resident macrophages express, on their surface, carbohydrate epitopes which undergo changes during their stimulation/activation as monitored by binding of125I labelledEvonymus europaea andGriffonia simplicifolia I-B4 lectins. Treatment of the stimulated macrophages with coffee bean -galactosidase abolished binding of the GS I-B4 isolectin and changed the binding pattern of theEvonymus lectin. The affinity (K a) ofEvonymus lectin for -galactosidase-treated macrophages decreased approximately 23-fold, from 1.25×108 M–1 to 5.5×106 M–1. Subsequent digestion of -galactosidase-treated macrophages with -l-fucosidase fromTrichomonas foetus, further reduced binding ofEvonymus lectin. Resident macrophages showed the same pattern ofEvonymus lectin binding, with the same affinity, as -galactosidase-treated, stimulated macrophages. These results, together with a consideration of the carbohydrate binding specificity of theEvonymus lectin which, in the absence of -d-galactosyl groups, requires -l-fucosyl groups for binding, indicate the presence, on resident macrophages, of glycoconjugates with terminal -l-fucosyl residues. It is also concluded that during macrophage stimulation/activation -d-galactosyl residues are added to this glycoconjugate and that they form part of the receptor forEvonymus lectin. The same glycoconjugate(s) is/are also expressed on the activated macrophage IC-21 cell line which exhibits the same characteristics as that of stimulated peritoneal macrophages, i.e., it contains -d-galactosyl end groups and is resistant to the action of trypsin. Both lectins were also specifically bound toCorynaebacterium parvum activated macrophages.Abbreviations BSA bovine serum albumin - GS I-B4 Griffonia simplicifolia I-B4 isolectin - PBS 0.01m phosphate buffer (pH 7.1) with 0.15m NaCl (unless stated otherwise this buffer contained 3mm azide and was free of divalent cations) - PMSF phenyl methane sulfonyl fluoride - TG thioglycollate brewers medium.  相似文献   

13.
Summary An X-ray diffraction analysis ofZ-l-Leu-Aib-Gly-l-Ile-l-Leu-OMe, containing the N-acylated tetrapeptide amide sequence-l-Leu-Aib-Gly-l-Ile-, showed that in the crystal state the carbonyl group preceding thel-Leu1 residue acts as the acceptor of two C=OH–N intramolecular H-bonds, which give rise to an-l-Leu1-Aib2-type-III' -turn and an-l-Leu1-Aib2-Gly3-l-Ile4--turn, respectively. A second (type-I') -turn encompasses the-Aib2-Gly3-sequence. This is the third type of folding motif known for that tetrapeptide sequence, considering also those already published for the C-terminal segment of the lipopeptaibol antibiotics trichodecenin I and trichogin A IV.  相似文献   

14.
Two types of nitrosoguanidine-induced rifampicin-resistant mutants ofNostoc muscorum were isolated and characterized. Compared with the wild type, the strainrif-1 (rif r het + nif + blu) revealed high growth rate, heterocyst frequency (10%–12%), nitrogenase activity, phycocyanin pigment, photosynthetic O2 evolution, and higher activities of phosphoribulokinase and Fd-NADP+-oxidoreductase. The heterocyst spacing pattern in the mutant was altered and did not respond to 7-azatryptophan, -2-thienylalanine, rifampicin, andl-methionine-dl-sulfoximine treatment. The second type of mutantrif r -2 (rif r het + nif ) did not show nitrogenase activity and was unable to grow on molecular nitrogen even under microaerobic conditions, although it produced heterocysts (6%–7%) under these conditions of incubation. The pattern of macromolecular synthesis, particularly of RNA and protein, the rate of acetylene reduction, and photosynthetic O2 evolution were not affected in the mutant strains with the treatment of drug. The characteristics of the mutants reflected the possibility of pleiotropic mutation and also suggested that therif marker is most likely associated in close genetic proximity with regulatory gene(s) ofhet andnif system.  相似文献   

15.
Summary Transport of the nucleoside analog cytosine-arabinoside (CAR) in transformed hamster cells in culture has been studied in conditions of minimal metabolic conversion. Uptake (zero-trans in) properties at 20°C over a limited range of CAR concentrations were characterized by aK m of 350 m and a maximal velocity (V) of 780 m·min–1 (V/K m =2.28 min–1). Equilibrium exchange at 20°C over a wider range of concentrations was best described by a saturable component with aK m of 500 m and av of 1230 m·min–1 (V/K m =2.26 min–1) and either a saturable component of highK m or a nonsaturable component ofk=0.3 min–1. For the saturable component, thev/K m values were similar in both procedures.CAR transport was inhibited by various metabolizable nucleosides. Uptake of some of these nucleosides was inhibited by CAR. CAR transport and uridine uptake were inhibited in a reversible but partially competitive fashion by high affinity probes like S-(p-nitrobenzyl-6-mercaptoinosine (NBMI) (K i <0.5nm) and in an irreversible fashion by SH reagents such as N-ethylmaleiimide (NEM). The organomercurialp-hydroxymercuribenzene sulfonate (pMBS) markedly stimulated transport of these nucleosides, but also markedly potentiated the inhibitory effects of either NBMI or NEM. These effects are interpreted either in terms of models which invoke allosteric properties or in terms of two transport systems which display distinct chemical susceptibilities to externally added probes.  相似文献   

16.
Summary Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20°C in the presence of [3H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na+ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 m and at 37°C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes,K m =0.12, 0.11 and 0.10 m andJ max=0.01, 0.02 and 0.03 pmol·l–1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts,K m =0.16 and 0.51 m andJ max=0.01 and 0.04 pmol·l–1 intracellular water after 15 and 30 min incubation times, respectively. At 20°C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence ofd-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein-(probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.  相似文献   

17.
Summary Pancreatic islet B cells depolarize and display trains of action potentials in response to stimulatory concentrations of glucose. Based on data from rodent islets these action potentials are considered to be predominantly Ca2+ dependent. Here we describe Na+-dependent action potentials and Na+ currents recorded from canine and human pancreatic islet B cells. Current-clamp recording using the nystatin perforated-patch technique demonstrates that B cells from both species display tetrodotoxin-sensitive Na+ action potentials in response to modest glucose-induced depolarization. In companion whole-cell voltage-clamp experiments on canine B cells, the underlying Na+ current displays steep voltage-dependent activation and inactivation over the range of –50 to –40 mV. The Na+ current is sensitive to tetrodotoxin block with aK 1=3.2nm and has a reversal potential which changes with [Na+] o as predicted by the Nernst equation. These results suggest that a voltage-dependent Na+ current may contribute significantly to action potential generation in some species outside the rodent family.  相似文献   

18.
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   

19.
Summary The current-voltage (I/V) technique was employed to investigate the different electrophysiological states of theChara plasmalemma and their interaction under a range of conditions. In K+ state the membrane became very permeable (conductances >20 S m 2) as [K+]0 increased to 10mm. As the cells were then easily damaged by the voltage-clamp procedures, it was difficult to determine the saturation K+ conductance. TEA (tetraethylammonium chloride) reversibly blocked the K+ channels, but had no effect on theI/V curve of the pump state, indicating that the K+ channels were not participating in this state. Acid pH0 (4.5) diminished the K+ conductance, but did not alter the response of the K+ channels to change in [K+]0. Alkaline pH0 (11.0) madeChara resting PD bistable: the PD either stayed near the estimatedE K and theI/V curve showed a negative conductance region typical of the K+ state, or it hyperpolarized and the near-linearI/V profile of the proton-permeable state was observed.  相似文献   

20.
Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID50 (0.3±0.1 m) for H2-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of DBDS is virtually the same as the ID50 (0.47±0.04 m) for H2-DIDS inhibition of red cell Cl flux, thus relating DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID50 for cytochalasin B modulation of DBDS is 0.1±0.2 m in good agreement withK D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na+,K+-ATPase, increases red cell Cl exchange flux in red cells by a factor of about two. This interaction indicates that the Na+,K+-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.  相似文献   

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