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1.
Summary The choline carrier of human erythrocyte membranes exists in distinguishable outward-facing and inward-facing conformations, and previous studies demonstrated that only the latter reacts with N-ethylmaleimide, producing an irreversible inhibition of transport. We now report experiments to determine the individual reaction rates for the two inward-facing forms: the free carrier and the complex. The pseudo-first-order rate constant for the complex with a substrate analog, di-n-butylaminoethanol, is found to be nearlydouble that for the free carrier, showing that the carrier conformation is altered following addition of a ligand (with 1mm N-ethylmaleimide at pH 6.8, 37°C, the constants are 0.57±0.05 min–1 and 0.33±0.02 min–1, respectively). Hence three different conformational states have been distinguished by experiment: (1) the inward-facing free carrier; (2) the inward-facing complex; and (3) the outward-facing carrier.  相似文献   

2.
A method is described, based on the kinetics of transport, for determining the equilibrium distribution of the carrier site on the inner and outer surfaces of the cell membrane, and this method is applied to the choline carrier of human erythrocytes. This method depends on measurement of flux ratios for both entry and exit, i.e., the transport rates of a low concentration of labeled substrate into a solution which contains either no substrate or a saturating concentration of unlabeled substrate. The concentrations of inward-facing and outward-facing carrier are found to be nearly equal, and therefore the 5-fold difference in choline affinity on the inner and outer surfaces of the membrane cannot be explained by an unequal carrier distribution. It is also shown that both reorientation and dissociation of the carrier-substrate complex are far more rapid than reorientation of the free carrier.  相似文献   

3.
Inhibition of choline transport in erythrocytes by n-alkanols   总被引:1,自引:0,他引:1  
The choline transport system of erythrocytes is reversibly inhibited by ethanol, n-butanol, n-hexanol, n-octanol, and n-decanol, but not by n-dodecanol. Each methylene group in the alkyl chain contributes 560 cal/mol to the free energy of binding at the inhibitory site. Inhibition results from the cooperative binding of two molecules of an alcohol, judging by the Hill coefficient n of 1.7-1.9. The mechanism of inhibition is noncompetitive, and the partition of the carrier between inward-facing and outward-facing forms is unaffected by the alcohols; it follows that the four main carrier forms, the inner and outer free carrier, and the inner and outer carrier-substrate complex, are equally susceptible to inhibition. Hexanol and decanol accelerate the reaction of N-ethylmaleimide with a thiol group in the inner carrier channel, but ethanol and butanol, at concentrations that inhibit transport by 70%, do not. The disproportionate effects on substrate transport and the N-ethylmaleimide reaction are most simply explained as the direct result of binding of alcohol molecules in different regions of the carrier, rather than as the indirect result of a disturbance in the structure of the lipid bilayer induced by the alcohols.  相似文献   

4.
Summary Under zero-trans conditions, the facilitated transport of choline across the erythrocyte membrane is limited by the rate of reorientation of the free carrier; as a result the pH dependence of this step can be investigated, independent of other steps in transport. It is found that as the pH declines (between 8.0 and 6.0) the rate of inward movement of the free carrier rises and the rate of outward movements falls, so that the partition of the free carrier increasingly favors the inward-facing form. When the pH of the cell interior and of the medium are varied independently, the partition responds to the internal but not the external pH. The membrane potential, which varies somewhat as the pH is altered, has no effect on the carrier partition. The analysis of the results indicates that the carrier mobility is dependent on an ionizing group of pK a 6.8, which is exposed on the cytoplasmic surface of the membrane in the inward-facing carrier; in the out-ward-facing carrier the ionizing group appears to be masked, in that its pK a is shifted downward by more than one unit. The observations can be explained by assuming that an ionizing group is located in the wall of a gated channel connecting the substrate site with the cytoplasmic face of the cell membrane.  相似文献   

5.
Summary The properties of the choline transport system are fundamentally altered in saline solution containing 5mm imidazole buffer instead of 5mm phosphate: (i) The system no longer exhibits accelerated exchange. (ii) Choline in the external compartment fails to increase the rate of inactivation of the carrier by N-ethylmaleimide. (iii) Depending on the relative concentrations of choline and imidazole, transport may be activated or inhibited. The maximum rates are increased more than fivefold by imidazole, but at moderate substrate concentrations activation is observed with low concentrations of imidazole and inhibition with high concentrations. (iv) The imidazole effect is asymmetric, there being a greater tendency to activate exit than entry. All this behavior is predicted by the carrier model if imidazole is a substrate of the choline carrier having a high maximum transport rate but a relatively low affinity, and if imidazole rapidly enters the cell by simple diffusion, so that it can add to carrier sites on both sides of the membrane. Addition at thecis side inhibits, and at thetrans side activates. According to the carrier model, asymmetry is a necessary consequence of the potassium ion gradient in red cells, potassium ion being another substrate of the choline system.  相似文献   

6.
Summary The substrate specificities on the inner and outer surfaces of the cell membrane have been compared by determining the relative affinities, inside and outside, of a series of choline analogs. The results of two different methods were in agreement: (1) the carrier distribution was determined in the presence of a saturating concentration of an equilibrated analog, using N-ethylmaleimide as a probe for the inward-facing carrier; (2) the degree of competition was measured between an equilibrated analog and choline in the external solution. The carrier sites are found to have markedly different specificities: the outer site is more closely complementary to the structure of choline than is the inner, and even a slight enlargement of either the trimethylammonium or hydroxyethyl group gives rise to preferential binding inside. It is also found that a nonpolar binding region, which is adjacent to the outer site, is absent from the inner site. As the transport mechanism involves the exposure of only one site at a time, first on one surface and then the other, it follows that an extensive reorganization of the structure of the substrate site may occur during the carrier-reorientation step, or alternatively that two distinct sites may be present, only one of which is exposed at a time.  相似文献   

7.
Summary Choline transport across the human erythrocyte membrane is irreversibly inhibited when N-ethylmaleimide (NEM) reacts with a carrier SH group which is located outside the substrate site, and which is exposed in the inward-facing form of the carrier but prevented from reacting in the outward-facing form. The location of the SH group with respect to the membrane has now been determined by studying the dependence of the NEM-alkylation rate on the intracellular and extracellular pH. The results show that the reactive SH group equilibrates with hydrogen ions in the cytoplasm, but is completely isolated from hydrogen ions in the external medium. With this added evidence it becomes possible to conclude that the SH group is located in the inner gated channel of the choline carrier.  相似文献   

8.
The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys148 is converted to a glycine residue. Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid. Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-β-D-galactopyranoside affords no protection against inactivation. The findings suggest that although cys148 is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose:proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity.  相似文献   

9.
The sodium dependent transport system for L-glutamate and L-aspartate localized in the apical part of rat enterocytes has previously been kinetically characterized (Prezioso, G., and Scalera, V. (1996). Biochim. Biophys. Acta 1279, 144–148). In this paper the mechanism by which the potassium cation specifically activates the L-glutamate–sodium cotransport process is investigated. Potassium has been found to act as an activator when it is present inside the membrane vesicles, while its presence outside is ineffective, and the effect is saturable. The kinetic parameters with respect to sodium and glutamate have been compared in the presence and in the absence of the activator. The results indicate that the ordered sodium–sodium glutamate mechanism is not altered by potassium, and that the activation is probably exerted on both the rate determining steps of the transport process. It is proposed that (1) a specific binding site for potassium is present on the inside hydrophilic part of the membrane carrier, (2) the binding of the effector accelerates the intramembrane rearrangement steps of both the disodium glutamate–carrier complex and the free carrier, (3) the affinity of the carrier is lowered with respect to sodium whereas it is increased for glutamate, and (4) K+ antiport is not performed by this carrier.  相似文献   

10.
Summary Evidence for a mobile membrane carrier mediating the uphill monosaccharide transport in the yeastRhodotorula gracilis is based on two types of observations: (1) Countertransport was found with14C-labelledd-xylose,l-xylose,l-rhamnose and withl-rhamnose in a cell suspension preincubated with unlabelledd-xylose. This finding indicates, moreover, that both the hexoses and the pentose share the same membrane carrier. (2) The mobility of occupied carrier molecules is higher than that of free carrier molecules. This conclusion has been drawn from: (a) comparison of the initial rates of uptake of a labelled sugar into cells preincubated in the absence and in the presence of unlabelled sugar; (b) comparison on the half-saturation constant of transport with the dissociation constant of the sugar-carrier complex; and (c) comparison of the initial rates of efflux of a labelled sugar into sugar-free and sugar-containing medium.  相似文献   

11.
Through the use of the protein reagents N-ethylmaleimide, p-chloromercuribenzenesulphonic acid and phenylarsine oxide, it is shown that in the scutellum of the germinating barley embryo, the transport of peptides, but not the transport of amino acids or glucose is specifically thiol-dependent. Furthermore, these essential thiol groups are shown to exist as redox-sensitive, vicinal-dithiols that lie at the substrate-binding sites of the peptide-transport proteins. The binding of N-ethylmaleimide to these dithiols is shown to be very fast, matching the kinetics of inhibition of peptide transport by this reagent. A technique for the specific labelling of the dithiols with N-ethyl[2,3-14C]maleimide is described, which allows the carrier proteins to be visualized at the scutellar epithelium using radioautography and permits calculation of the approximate amount of peptide-transport protein present per scutellum. In related studies, the importance of arginyl and histidyl residues to both amino-acid and peptide transport is shown, although other residues, e.g. carboxyl ligands do not seem to be critically involved.Abbreviations Ala alanine - Gly glycine - Leu Leucine - NEM N-ethylmaleimide - PAO phenylarsine oxide - PCMBS p-chloromercuribenzenesulphonic acid - Phe phenylalanine  相似文献   

12.
Simulation shows that the four-state mobile carrier model for sugar transport in which the asymmetry arises from unequal rate constants of inward and outward translation of the free-carrier and carrier-sugar complex, does not fit with the observed data for pre-steady-state uptake recently obtained by A.G. Lowe and A.R. Walmsley ((1987) Biochim. Biophys. Acta 903, 547–550). The main reason for this discrepancy is that pre-steady-state fluxes are determined mainly by the dissociation constants Ks of glucose and maltose for the external sites, rather than the Km (zero-transoi) of glucose and the Ki of maltose. The data are also inconsistent with other forms of asymmetric carrier but are fairly consistent with a symmetrical carrier with high-affinity sites for -glucose or with a fixed site carrier model.  相似文献   

13.
The amino acid transport across the plasmalemma of Riccia fluitans rhizoid cells has been further characterized by means of current-voltage I?V) analysis. On the basis of two cyclic transport models which include six different carrier states, the question is raised, whether the electrochemical pH-gradient drives a negatively charged carrier or a positively charged alanine-proton-carrier complex across the membrane. I?V analysis shows that (1) the typical I?V characteristic of l-alanine transport follows a sigmoid curve, (2) maximal accumulation of l-alanine within the cytoplasm is reached after about 1 hour, (3) the electrically accessible cytoplasmic l-alanine concentration is limited to about 20 mM, and (4) the steady-state saturation current depends directly on external l-alanine concentration. It is concluded that (a) these results are consistent with the predictions of the models for a negatively charged carrier, and (b) that the rate-limiting step involves the translocation of the ternary complex.  相似文献   

14.
Inhibition of hexose transport by N-ethylmaleimide was studied with regard to alkylation of different types of sulfhydryl group on the hexose carrier of the human erythrocyte. Uptake of 3-O-methylglucose was progressively and irreversibly inhibited by N-ethylmaleimide, with a half-maximal effect at 10-13 mM. A sulfhydryl group known to exist on the exofacial carrier was not involved in transport inhibition by N-ethylmaleimide, since reversible protection of this group by the impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) had no effect on the ability of N-ethylmaleimide to inhibit transport, or on its ability to decrease the affinity of the exofacial carrier for maltose. Nevertheless, the exofacial sulfhydryl was quite reactive with N-ethylmaleimide, since it was possible using a differential labeling technique to specifically label this group in protein-depleted ghosts with a half-maximal effect at 0.3 mM N-[3H]ethylmaleimide, and to localize it to the Mr 19,000 tryptic carrier fragment. Transport inhibition by N-ethylmaleimide correlated best with labeling of a single cytochalasin B-sensitive internal sulfhydryl group on the glycosylated Mr 23,000-40,000 tryptic fragment of the carrier, which was half-maximally labeled at about 4 mM reagent. Whereas N-ethylmaleimide readily alkylates the exofacial carrier sulfhydryl, it inhibits transport by reacting with at least one internal carrier sulfhydryl located on the glycosylated tryptic carrier fragment.  相似文献   

15.
1. The dependence of V and V/K(m) for threonine transport into Trypanosoma brucei upon the external concentration of H(+) was studied. 2. Two ionizing groups, the alpha-amino group of the substrate and a group at the substrate-binding site of the carrier, were found to influence the observed kinetic behaviour of transport. 3. The pK of the group at the substrate-binding site on the free carrier was found to be 6.95 at 30 degrees C and to be temperature-dependent; its heat of ionization was -63.8kJ, which is outside the range for most proton dissociations and suggests a significant contribution from some other source, possibly the remainder of the carrier or the membrane environment. 4. Binding of substrate caused the pK of its alpha-amino group to shift to a higher value, whereas that of the carrier group shifted to a lower value (6.65 at 30 degrees C). 5. The ionic interaction between substrate and carrier appeared to be involved in the stabilizing of the protonated substrate and the species of the carrier-substrate complex required for the membrane-translocation step. 6. The same ionic species of carrier-substrate complex is required for both substrate dissociation and translocation of the substrate through the membrane. 7. H(+) symport or antiport did not occur during threonine uptake.  相似文献   

16.
A functional model for the aspartate/glutamate carrier of the inner mitochondrial membrane was established based on a kinetic evaluation of this transporter. Antiport kinetics were measured in proteoliposomes that contained partially purified carrier protein of definite transmembrane orientation (Dierks, T. and Kr?mer, R. (1988) Biochim. Biophys. Acta 937, 122-126). Bireactant initial velocity analyses of the counterexchange reaction were carried out varying substrate concentrations both in the internal and the external compartment. The kinetic patterns obtained were inconsistent with a pong-pong mechanism; rather they demonstrated the formation of a ternary complex as a consequence of sequential binding of one internal and one external substrate molecule to the carrier. Studies on transport activity in the presence of aspartate and glutamate in the same compartment (formally treated as substrate inhibition) clearly indicated that during exchange only one form of the carrier at either membrane surface exposes its binding sites, for which the two different substrates compete. In the deenergized state (pH 6.5) both substrates were translocated at about the same rate. Aspartate/glutamate antiport became asymmetric if a membrane potential was imposed, due to the electrogenic nature of the heteroexchange resulting from proton cotransport together with glutamate. Investigation of the electrical properties of aspartate/aspartate homoexchange led to the conclusion that the translocating carrier-substrate intermediate exhibits a transmembrane symmetry with respect to the (negative) charge, which again only is conceivable assuming a ternary complex. Thus, an antiport model is outlined that shows the functional complex of the carrier with two substrate molecules bound, one at either side of the membrane. The conformational change associated with the transition of both substrate molecules across the membrane then occurs in a single step. Furthermore the model implicates a distinct proton binding site, which is derived from the different influence of H+ concentration observed on transport affinity and transport velocity, respectively, when glutamate is used as a substrate.  相似文献   

17.
Summary The involvement of a histidyl residue in the binding or translocation step was investigated in the malate carrier at the tonoplast of Catharanthus roseus cells. The transport rate was strongly stimulated when the pH of the incubation medium was decreased from pH 7.0 to 5.0. The histidine-specific reagent diethylpyrocarbonate (DEPC) efficiently inhibited the activity of the malate carrier. Inhibition developed rapidly and was completed after 5 min at a concentration of 2 mM DEPC. The original substrate, malate, partially protected the carrier from inactivation by DEPC. Other organic acids (citrate, quinate) which are known to affect the malate transport of isolated vacuoles or tonoplast vesicles also showed protective properties. Inhibition of malate transport on tonoplast vesicles can also be achieved by photooxidation in the presence of the dye Rose Bengal. Malate also proved to protect against inactivation.The results strongly support the notion that a histidyl residue(s) is involved either in the binding or translocation of malate and that the protonation of the histidyl residue is essential to provide a high rate of malate transport.This research was supported by the Centre National de la Recherche Scientifique and by a grant from the European Community (BRIDGE program). K.-J. Dietz acknowledges support by the Jubiläumsstiftung der Julius-Maximilians-Universität Würzburg, which made the stay in Toulouse possible, and the Sonderforschungsbereich 176.  相似文献   

18.
Summary The sidedness of phloretin binding to the glucose carrier has been determined by comparing the type of inhibition produced in zerotrans entry and zerotrans exit experiments. Initial rates of zerotrans entry were measured by the method of R.D. Taverna and R.G. Langdon (Biochim. Biophys. Acta 298:412–421, 1973), which involves pink ghosts loaded with glucose oxidase; this obviates the problem of rapid substrate accumulation inside the cells. With phloretin equilibrated across the membrane, the inhibition of entry was competitive, and the inhibition of exit noncompetitive. The experimental procedures were validated by showing that the inhibition by cytochalasin B, known to bind inside but not outside, was noncompetitive in entry and competitive in exit, as predicted. It was also demonstrated that even after pre-incubation of the cells with a relatively high concentration of phloretin, the phloretin adsorbed in the membrane did not significantly alter the rate of carrier reorientation. The results show that the outward-facing form of the glucose carrier, but not the inward-facing form, bears a phloretin binding site; thus phloretin, as well as cytochalasin B, is bound asymmetrically, phloretin outside and cytochalasin B inside.  相似文献   

19.
The relationships between structure, affinity and transport activity in the choline transport system of erythrocytes have been investigated in order to (i) explore the nature of the carrier site and its surroundings, and (ii) determine the dependence of the carrier reorientation process on binding energies and steric restraints due to the substrate molecule. Affinity constants and maximum transport rates for a series of trialkyl derivatives of ethanolamine were obtained by a method that involves measuring the trans effect of unlabeled analogs upon the movement of radioactive choline. The main conclusions are as follows: (1) An analysis of transport kinetics shows that the affinity constants determined experimentally differ from the actual dissociation constants in a predictable way. The better the substrate, the higher the apparent affinity relative to the true value, whereas the affinity of non-transported inhibitors is underestimated by a constant factor. (2) The carrier-choline complex undergoes far more rapid reorientation (translocation) than the free carrier. (3) The carrier imposes a strict upper limit upon the size of a substrate molecule that can participate in the carrier reorientation process; this limit corresponds to the choline structure. A smaller substrate such as tetramethylammonium, despite relatively weak binding forces, is unhindered in its translocation, suggesting that a carrier conformational change, dependent upon substrate binding energy, is not required for transport. (4) Small increases in the size of the quaternary ammonium head, as in triethylcholine, sharply lower affinity, consistent with a high degree of specificity for the trimethylammonium group. (5) Lengthening the alkyl substituent in derivatives of dimethyl- and diethylaminoethanol causes a regular increase in affinity, suggestive of unspecific hydrophobic bonding in a region very near the substrate site.  相似文献   

20.
The present study involves molecular docking, molecular dynamics (MD) simulation studies, and Caco‐2 cell monolayer permeability assay to investigate the effect of structural modifications on PepT1‐mediated transport of thyrotropin releasing hormone (TRH) analogs. Molecular docking of four TRH analogs was performed using a homology model of human PepT1 followed by subsequent MD simulation studies. Caco‐2 cell monolayer permeability studies of four TRH analogs were performed at apical to basolateral and basolateral to apical directions. Inhibition experiments were carried out using Gly‐Sar, a typical PepT1 substrate, to confirm the PepT1‐mediated transport mechanism of TRH analogs. Papp of the four analogs follows the order: NP‐1894 < NP‐2378 < NP‐1896 < NP‐1895. Higher absorptive transport was observed in the case of TRH analogs, indicating the possibility of a carrier‐mediated transport mechanism. Further, the significant inhibition of the uptake of Gly‐Sar by TRH analogs confirmed the PepT1‐mediated transport mechanism. Glide docking scores of all the four analogues were in good agreement with their transport rates, suggesting the role of substrate binding affinity in the PepT1‐mediated transport of TRH analogs. MD simulation studies revealed that the polar interactions with amino acid residues present in the active site are primarily responsible for substrate binding, and a downward trend was observed with the increase in bulkiness at the N‐histidyl moiety of TRH analogs. Copyright © 2014 John Wiley & Sons, Ltd.  相似文献   

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