共查询到20条相似文献,搜索用时 31 毫秒
1.
George A. Vidaver Sara L. Shepherd Joyce B. Lagow Karen J. Wiechelman 《生物化学与生物物理学报:生物膜》1976,443(3):494-514
The influence of a Donnan effect on the transport of glycine by hemolysed and restored pigeon red cells was examined. The Donnan effect was produced by replacing Cl? with 2,4-toluenedisulfonate or glutamate. The effects of the associated membrane potential and inside-outside pH difference on glycine entry and exit rates were examined. The effects of pH on entry and exit rates in the absence of a Donnan effect were also examined.In the absence of a Donnan effect, Na+-dependent glycine entry requires the protonated form of a group with a pKapp of 7.9 and the depronated form of another group with a pKapp of 6.8. Neither of these are required for exit but the deprotonated form of a group(s) with a pKapp of 6.2 is required. The pK 7.9 group and pK 6.2 group probably react with H+ at the inner face of the membrane and the pK 6.8 group probably reacts at the outer face.The V for glycine entry was determined for cells with their Cl? largely replaced by toluenedisulfonate and without such replacement. Between pH 6.1 and 7, the ratio of the respective V values, VT/VCl, was 1.5–1.7. VT/VCl rose above pH 7 to near 4 at pH 8.3. At pH 6.9, with glutamate replacing cell Cl?, the analogous ratio (VGlu/VCl) was 1.7. The increase of VT/VCl above pH 7 could be quantitatively accounted for by the increase in cell [H+]/medium [H+] caused by the Donnan effect together with the assumption that the pK 7.9 group reacts with H+ at the inner face of the membrane.When cell Cl? was replaced by toluenedisulfonate or glutamate there was a drop in the term in the glycine Km describing Na+ dependence of glycine entry. When cell Cl? was replaced by toluenedisulfonate there was a rise in the Na+-independent term in the glycine entry Km. By replacing varying amounts of cell Cl? with either toluenedisulfonate or glutamate, plots were obtained of entry rates vs. the cell [Cl?]/medium [Cl?] ratio consistent with the assumption that the Donnan-induced membrane potential acts on a “moving” charge. Glycine exit was only slightly accelerated by trans-toluenedisulfonate. The ratio, exit rate into toluenedisulfonate medium/exit rate into Cl? medium rose with decreasing pH. This rise could be accounted for by a Donnan-induced inside-outside pH difference which affects a pKapp 6.2 group reacting with internal H+.The observed influences of the Donnan effect on V(glycine entry), on both components of Km(glycine entry), on the shape of the plot of glycine entry rate vs. the cell [Cl?]/medium [Cl?] ratio and on glycine exit all fit the assumptions that when the empty porter reorients, one unit of negative charge accompanies it “across” the membrane and that no other steps involve charge movement.The properties of the system seem inconsistent with a translational (“ferry boar”) mobile carrier. 相似文献
2.
Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles
The sodium-dependent entry of proline and glycine into rat renal brushborder membrane vesicles was examined. The high Km system for proline shows no sodium dependence. The low Km system for glycine entry is strictly dependent on a Na+ gradient but shows no evidence of the carrier system having any affinity for Na+. The low Km system for proline and high Km system for glycine transport appear to be shared. Both systems are stimulated by a Na+ gradient and appear to have an affinity for the Na+. The effect of decreasing the Na+ concentration in the ionic gradient is to alter the Km for amino acid entry and, at low Na+ concentrations, to inhibit the V for glycine entry. 相似文献
3.
M E Tischler J Pachence J R Williamson K F La Noue 《Archives of biochemistry and biophysics》1976,173(2):448-461
In order to study the mechanism of the glutamate-aspartate translocator, rat liver mitochondria were loaded with either glutamate or aspartate. In the presence of ascorbate plus tetramethyl-p-phenylenediamine as an electron donor at the third energy conservation site, exchange of external glutamate for matrix aspartate is highly favored over the reverse exchange. In the absence of an energy source, although the asymmetry of the exchange rates is much smaller, it is still observable. Further studies have shown that the proton uptake accompanying influx of glutamate in exchange for aspartate efflux occurs by protonation of a group on the carrier (pK = 7.9) at the external side of the inner mitochondrial membrane, followed by deprotonation at the matrix surface. It is postulated that glutamate binds to the protonated form of the carrier and aspartate to the deprotonated form. Because of the alkaline pK, aspartate efflux is inhibited with increased matrix [H+] due to limitation of the availability of deprotonated carrier for aspartate binding. For the reverse exchange, aspartate uptake is inhibited by increasing external [H+]. Thus the rate of aspartate uptake by mitochondria is apparently impeded both by a proton motive force (Δp) unfavorable to entry of ions with net negative charge as well as by the small proportion of deprotonated carrier at the outer surface of the membrane. This conclusion is further illustrated by inhibition of the aspartate-aspartate exchange with increased [H+] and by addition of an energy source. The glutamate-glutamate exchange, however, showed a slight stimulation by increased [H+] and was unaffected by the energy state.The model initially proposed for the carrier, in which a neutral glutamate-carrier complex exchanges for a negatively charged aspartate-carrier complex, is tested further. Glutamate uptake was noncompetitively inhibited by external aspartate, which indicates that aspartate and glutamate bind to separate forms of the carrier. Intramitochrondrial glutamate at a concentration of 18 mm, however, had no effect on aspartate efflux. Arrhenius plots for the glutamate-aspartate and aspartate-glutamate exchanges were linear over the range of temperatures tested (1–35 °C and 5–25 °C, respectively) and provided an average value of 14.3 kcal/mol for the energy of activation. In addition, there appear to be two pools, exchangeable and nonexchangeable, of matrix aspartate available to the translocator, since extramitochondrial radiolabeled aspartate can equilibrate only with unlabeled matrix aspartate at low aspartate loading (1–2 nmol of aspartate/mg of protein). The physiological significance of the data is discussed. 相似文献
4.
The Hexose-Proton Cotransport System of Chlorella : pH-Dependent Change in Km Values and Translocation Constants of the Uptake System 总被引:13,自引:1,他引:12
下载免费PDF全文
![点击此处可从《The Journal of general physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The proton concentration in the medium affects the maximal velocity of sugar uptake with a Km of 0.3 mM (high affinity uptake). By decreasing the proton concentration a decrease in high affinity sugar uptake is observed, in parallel the activity of a low affinity uptake system (Km of 50 mM) rises. Both systems add up to 100%. The existence of the carrier in two conformational states (protonated and unprotonated) has been proposed therefore, the protonated form with high affinity to 6-deoxyglucose, the unprotonated form with low affinity. A plot of extrapolated Vmax values at low substrate concentration versus proton concentration results in a Km for protons of 0.14 µM, i.e. half-maximal protonation of the carrier is achieved at pH 6.85. The stoichiometry of protons cotransported per 6-deoxyglucose is close to 1 at pH 6.0–6.5. At higher pH values the stoichiometry continuously decreases; at pH 8.0 only one proton is cotransported per four molecules of sugar. Whereas the translocation of the protonated carrier is strictly dependent on sugar this coupling is less strict for the unprotonated form. Therefore at alkaline pH a considerable net efflux of accumulated sugar can occur. The dependence of sugar accumulation on pH has been measured. The decrease in accumulation with higher pH values can quantitatively be explained by the decrease in the amount of protonated carrier. The properties of the unprotonated carrier resemble strikingly the properties of carrier at the inner side of the membrane. The inside pH of Chlorella was measured with the weak acid 5,5-dimethyl-2, 4-oxazolidinedion (DMO). At an outside pH of 6.5 the internal pH was found to be 7.2. To explain the extent of sugar accumulation it has to be assumed that the membrane potential also contributes to active sugar transport in this alga. 相似文献
5.
C. S. Ramarao S. R. Acharya K. S. Krishnan U. W. Kenkare 《Journal of biosciences》1987,11(1-4):119-135
Preparations having properties resembling those of synaptosomes have been isolated from whole fly homogenates ofDrosophila melanogaster using ficoll gradient floatation technique. These have been characterized by marker enzymes and electron microscopy and binding of muscarinic antagenist3H Quinuclidinyl benzilate. An uptake system for neurotransmitter, ã-Aminobutyric acid has been demonstrated in these preparations. A high affinity uptake system for L-glutamate has also been studied in these subcellular fractions. This uptake of glutamate is transport into an osmotically sensitive compartment and not due to binding of glutamate to membrane components. The transport of glutamate has an obligatory requirements for either sodium or potassium ions. Kinetic experiments show that two transport systems, withK m values 0.33 X 10-6M and 2.0 X 10-6M, respectively, function in the accumulation of glutamate. ATP stimulates lower affinity transport of glutamate. Inhibition of glutamate uptake by L-aspartate but not by phenylalanine and tyrosine indicates that a common carrier mediates the transport of both glutamate and aspartate. β-N-oxalyl-L-β β-diamino propionic acid and kainic acid, both inhibitors of glutamate transport in mammalian brain preparations, strongly inhibited transport of glutamate inDrosophila preparations Comparison with uptake of ã-aminobutyric acid and glutamate in isolated larval brain is presented to show that the synaptosome-like preparations we have isolated are rich in central nervous system derived structures, and presynaptic endings from neuromuscular junctions. 相似文献
6.
Alcohols inhibit the exchange transport of glucose in human erythrocytes. Comparing the inhibition by monohydroxy-alcohols, which have different distribution coefficients between medium and membrane, shows that the degree of inhibition depends mainly upon the concentration of the alcohol in the membrane. 1-butanol exerts a mixed-type inhibition; Vmax decreases and Km increases. Since also the Km of the equilibrium transport increases upon the addition of the alcohol, the changes in the Km of exchange transport cannot be attributed solely to the differently affected mobilities of the loaded and free carrier, but the affinity of glucose to the transport system is reduced. The transport system can bind two alcohol molecules. With one alcohol molecule bound the affinity of the transport system for the second alcohol molecule already increases. The nature of the bond of the alcohols to the transport system is discussed and possible explanations for the cooperative effect upon the binding of the second alcohol molecule are offered. 相似文献
7.
Suspensions of 2 to 5% rat thymocytes were incubated at 35 °C in buffered balanced salt solution (pH 7.3) with lactate and β-hydroxybutyrate as fuels. The dependence of 3-O-[Me-3H]methylglucose influx on external and internal 3-O-methylglucose concentrations was studied. Entry was almost rectilinear during the first minute. From the dependence of methylglucose entry (into sugar-free cells) on external methylglucose concentration, we judged the entry Km to be about 7.7 mm and the entry V to be about 0.64 μmol · min?1 · (ml of packed cell volume)?1. Methylglucose inside the cell enhanced influx, hence equilibrium exchange was faster than entry. The dependence of equilibrium exchange on methylglucose concentration (inside and outside being equal) indicated a Km of about 25 mm and a V of about 2.1 μmol · (min)?1 · (ml of cell volume)?1. This effect of internal sugar indicated that entry into sugar-free cells is limited mainly by the return of empty carrier to the outside surface and that loading the carrier on the inside enhances its outward mobility. The Km and V for influx into cells containing 21 mm methylglucose were 5.9 mm and 1.17 μmol · min?1 · (ml of packed cells)?1. The effect of 21 mm internal sugar on lowering the influx Km from about 7.7 mm to about 6 mm was reproducible and contributed to the evaluation of the constants of the transport rate law. It indicated that loading of the carrier at the external surface reduces its mobility, in contrast to the effect of loading on the inside. Mechanical explanations for this behavior are discussed. 相似文献
8.
V. N. Samartsev M. V. Dubinin S. I. Adakeeva S. R. Rybakova E. I. Marchik 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2014,8(3):253-261
The effect of ion fluxes across the inner membrane on calcium-independent uncoupling activity of palmitic acid was investigated in experiments on rat liver mitochondria energized by the oxidation of succinate. The following compounds were used as the inductors of ion fluxes: the K+/H+ antiporter nigericin causing transformation of ΔpH into electrical potential difference (Δψ) across the inner membrane; tetraphenylphosphonium (TPP+) that freely crosses phospholipid membranes; protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) that induces a flow of H+ from the intermembrane space into the matrix and reduces Δψ and ΔpH. It was found that nigericin at a concentration of 20 nM, which causes an increase in maximal Δψ, partially inhibits the ability of palmitic acid to reduce Δψ and stimulates mitochondrial respiration. A specific inhibitor of the ATP/ADP antiporter (carboxyatractylate) and a substrate of the aspartate/glutamate antiporter (glutamate) increase Δψ and partially inhibit mitochondrial respiration in the presence of palmitic acid. Under these conditions, 10 μM cyclosporin A also inhibits respiration but has no effect on Δψ. The specific uncoupling activity of palmitic acid (V U) and its specific components that characterize participation of the ATP/ADP antiporter (V Catr), aspartate/glutamate antiporter (V Glu), and cyclosporin-A-sensitive system (V CsA) in the palmitic acid-induced uncoupling were estimated. It was shown that nigericin substantially reduces V U, V Catr and V Glu but increases V CsA. TPP+ at a concentration of 20 μM increases V U and V Glu, does not affect V Catr and reduces V CsA. FCCP at concentrations of 20 and 40 nM reduces Δψ by not more than 17% but does not affect V U, V Catr, V Glu and V CsA. The results suggest that the calcium-independent uncoupling effect of palmitic acid in liver mitochondria is caused by the return of protons to the matrix with participation of ADP/ATP and aspartate/glutamate antiporters and owing to activation of cyclosporin A-sensitive electron transport along the respiratory chain without affecting Δψ. The induced ion fluxes across the inner mitochondrial membrane can be considered as a factor of the calcium-independent regulation of uncoupling activity of palmitic acid in liver mitochondria with participation of the ADP/ATP and aspartate/glutamate antiporters and of the cyclosporin A-sensitive electron transport system. 相似文献
9.
10.
Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na+ gradient across the membrane was imposed. The maximum effect of Na+ on the transport was achieved at a concentration of about 40 mM, while the apparent Km for Na+ was approximately 8 mM. On the other hand, Km for glutamate in the presence of 50 mM Na+ was about 8 μM. Increasing the concentration of Na+ resulted in a decrease in Km for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na+ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K+-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent Km for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na+ dependent and the other is H+ dependent. 相似文献
11.
Glutamate transport by the neuronal excitatory amino acid carrier (EAAC1) is accompanied by the coupled movement of one proton across the membrane. We have demonstrated previously that the cotransported proton binds to the carrier in the absence of glutamate and, thus, modulates the EAAC1 affinity for glutamate. Here, we used site-directed mutagenesis together with a rapid kinetic technique that allows one to generate sub-millisecond glutamate concentration jumps to locate possible binding sites of the glutamate transporter for the cotransported proton. One candidate for this binding site, the highly conserved glutamic acid residue Glu-373 of EAAC1, was mutated to glutamine. Our results demonstrate that the mutant transporter does not catalyze net transport of glutamate, whereas Na(+)/glutamate homoexchange is unimpaired. Furthermore, the voltage dependence of the rates of Na(+) binding and glutamate translocation are unchanged compared with the wild-type. In contrast to the wild-type, however, homoexchange of the E373Q transporter is completely pH-independent. In line with these findings the transport kinetics of the mutant EAAC1 show no deuterium isotope effect. Thus, we suggest a new transport mechanism, in which Glu-373 forms part of the binding site of EAAC1 for the cotransported proton. In this model, protonation of Glu-373 is required for Na(+)/glutamate translocation, whereas the relocation of the carrier is only possible when Glu-373 is negatively charged. Interestingly, the Glu-373-homologous amino acid residue is glutamine in the related neutral amino acid transporter alanine-serine-cysteine transporter. The function of alanine-serine-cysteine transporter is neither potassium- nor proton-dependent. Consequently, our results emphasize the general importance of glutamate and aspartate residues for proton transport across membranes. 相似文献
12.
Dicarboxylate transport in maize mesophyll chloroplasts 总被引:7,自引:0,他引:7
Evidence is presented for high rates of carrier-mediated dicarboxylate anion transport in maize mesophyll chloroplasts. Radioactively labeled malate is transported across the chloroplast envelope leading to accumulation in the stroma. Malate in the stroma will exchange for external malate, oxaloacetate, glutamate, aspartate, and oxoglutarate. At 4 °C the V of malate uptake is 50 μmol·h?1·mg Chl?1 and the Km for malate is 0.5 mm. Oxaloacetate competitively inhibits malate uptake with a Ki estimated to be 0.3 mm. The temperature dependence of malate uptake indicates an activation energy of 12 kcal/mol, and extrapolation using this value gives a rate of transport at 30 °C of approximately 300 μmol·h?1·mg Chl?1. This rate approximates the rates of photosynthetic malate production by these chloroplasts. 相似文献
13.
《Experimental mycology》1983,7(3):233-240
GerminatingPhycomyces spores accumulated exogenous glutamine, glutamate, asparagine, and aspartate by a carrier-mediated active process with aKm in the millimolar range. The transport activity of heat-activated spores was much higher than in dormant spores. This increase was not only due to newly synthesized carrier molecules since cycloheximide inhibited the increase only partly. Amino acid transport activity was greatly enhanced in the presence of exogenous glucose. Besides its effect as energy source, glucose also seems to have a more indirect role in activated spores since its effect could be mimicked by 6-deoxy-d-glucose. Once transported into the spores, glutamate and aspartate were quickly metabolized suggesting also that the endogenous amino acids could serve as internal substrates. 相似文献
14.
Cells dissociated from the R3230AC mammary adenocarcinoma from intact and diabetic rats were examined for insulin binding and glucose transport. The Kd for insulin binding, ~ 10?10 M, was similar in all tumors studied. However, the apparent number of receptor sites per cell increased in cells from diabetic rats. Kinetic analysis of 3-0-methyl glucose (3-OMG) entry showed both diffusional and passive carrier characteristics. Insulin (4 × 10?9 M) in vitro did not affect diffusional entry, whereas the hormone altered the passive carrier system, as reflected by an increase in Km and Vmax. Insulin decreased initial velocity of glucose transport at 4–6 mM glucose levels but increased initial velocity of glucose transport at 20 mM glucose. An explanation of the role of insulin on tumor growth in vivo from effects on glucose transport in vitro is proposed. 相似文献
15.
Robert Roskoski Nancy Rauch Laura M. Roskoski 《Archives of biochemistry and biophysics》1981,207(2):407-415
To prepare membrane vesicles, nerve terminal preparations (synaptosomes) isolated from rat cerebral cortex were first subjected to hypotonic lysis. After collecting the membranes contained in this fraction by centrifugation, membrane vesicles were then reconstituted during incubation in a potassium salt solution at 37 °C. The transport of glutamate, aspartate, or γ-aminobutyric acid (GABA) was measured by transferring vesicles to 10 vol of 0.1 m NaCl solution containing the radioactive substrate. Transport was temperature dependent and exhibited saturation kinetics with an apparent Km of 2.5 μm. The rates and extent of l-glutamate and l-aspartate uptake were equivalent and were greater than those for GABA. Valinomycin increased the rate of uptake of each of these substances suggesting a role for an electrogenic component in transport. Consonant with this notion, external K+ and Rb+ decreased uptake of all three compounds. External thiocyanate also increases the rate of glutamate, aspartate, and GABA transport. Uptake of these neuroactive amino acids was absolutely dependent on external Na+; no other monovalent cation tested substitutes for it. Gramicidin D and nigericin inhibit glutamate transport by abolishing both the Na+ and K+ gradients. Monensin inhibits uptake by selectively dissipating the Na+ gradient. For both glutamate and GABA transport, the Na+ and K+ gradients are synergistic and not additive. 相似文献
16.
Inga H?nelt Sonja Jensen Dorith Wunnicke Dirk Jan Slotboom 《The Journal of biological chemistry》2015,290(26):15962-15972
GltPh from Pyrococcus horikoshii is a homotrimeric Na+-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na+ and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na+ binds with low affinity to the apoprotein (Kd 120 mm), with a particularly low kon value (5.1 m−1s−1). At least two sodium ions bind before aspartate. The binding of Na+ requires a very high activation energy (Ea 106.8 kJ mol−1) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na+ concentration present. Binding of aspartate was not observed in the absence of Na+, whereas in the presence of high Na+ concentrations (above the Kd for Na+) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (kon of 1.4 × 105
m−1s−1), with low Ea and Q10 values (42.6 kJ mol−1 and 1.8, respectively). We conclude that Na+ binding is most likely the rate-limiting step for substrate binding. 相似文献
17.
Transport of phosphate has been studied in subconfluent monolayers of LLC-PK1 cells. It was found that this transport system shows similar characteristics to those observed in the kidney. Uptake of phosphate is mediated by a Na+-dependent, substrate-saturable process with an apparent Km value for phosphate of 96 ± 15 μmol/1. Kinetic analysis of the effect of Na+ indicated that at (pH 7.4) two sodium ions are cotransported with one HPO42? ion (Hill coefficient 1.5) with an apparent Km value for sodium of 56 mmol/l. Pi uptake is inhibited by metabolic inhibitors (ouabain and FCCP). In the pH range of 6.6 of 7.4 Pi uptake rate does not change significantly, indicating that both the monovalent and the divalent form of phosphate are accepted by the transport system. It is suggested that phosphate is transported by LLC-PKi cells together with sodium (2 Na+ :1 HPO42?) in an electroneutral manner down a favourable sodium gradient. 相似文献
18.
Anion exchanger 1 (AE1 or band 3) is responsible for Cl−-HCO3− exchange on erythrocyte membrane. Previously, we showed that band 3 is fixed in an inward-facing conformation by specific modification of His 834 with DEPC, resulting in a strong inhibition of its anion transport activity. To clarify the physiological role of His 834, we evaluated the sulfate transport activities of various band 3 mutants: different mutants at His 834 and alanine mutants of peripheral residues around 834 (Lys 829-Phe 836) in yeast cell membranes. The Km values of the His 834 mutants were 4-10 times higher than that of the wild type, while their Vmax values were barely lower than that of wild type. Meanwhile, the Km values of the peripheral alanine mutants were only slightly increased. These data suggest that His 834 is critically important for the efficient binding of sulfate anion, but not for the conformational change induced by substrate binding. 相似文献
19.
Kinetic analyses of calcium movements in cell cultures. 3. Effects of calcium and parathyroid hormone in kidney cells 总被引:8,自引:0,他引:8
A B Borle 《The Journal of general physiology》1970,55(2):163-186
Calcium compartments and fluxes were measured by kinetic analyses in kidney cell suspensions in a three-compartment closed system. The fast phase influx and compartment size increase linearly with the medium calcium and the half-time of exchange is only 1.3 min which suggests that the fast component is extracellular. The slow phase compartment rises linearly from 0.1 to 0.5 mmole calcium/kg cell water when the medium calcium is raised from 0.02 to 2.5 mM. The slow phase calcium influx exhibits the pattern of saturation kinetics with a V
max of 0.065 µµmole cm-2 sec-1 and a Km of 0.3 mM indicating that it is a carrier-mediated transport process. PTH has no effect on the fast phase of calcium influx, but increases both calcium influx and the calcium pool size of the slow component. The maximum effect is obtained at medium calcium concentration of 1.3 mM. Below 0.3 mM extracellular calcium, the effects of the hormone cannot be demonstrated. PTH increases the V
max of calcium influx from 0.065 to 0.128 µµmole cm-2 sec-1 while the Km rises from 0.3 to 1.15 mM. These findings suggest that PTH increases the translocation of the calcium-carrier complex across the membrane and not the carrier concentration or its binding affinity for calcium. 相似文献
20.
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. 相似文献