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1.
(1) A flow-tube apparatus suited for measurement of rapid efflux of sugars from adipocytes is described. (2) Due to heterogeneity of fat cell populations, a conventional analysis of the time-course of net efflux of 3-O-methylglucose based on the integrated rate equation can produce gross errors in estimates of kinetic parameters. (3) The half-saturation constant and maximum transport capacity for 3-O-methylglucose transport were found to be about 3-fold higher for net efflux than for equilibrium exchange flux, both in insulin-stimulated and non-stimulated adipocytes. This suggests asymmetric kinetic parameters for 3-O-methylglucose transport.  相似文献   

2.
The kinetic parameters of the sugar transport in avian erythrocytes were evaluated under aerobic and anaerobic conditions. In anaerobic cells, transport measurements with 3-O-[14C] methylglucose resulted in a set of similar dissociation-like constants. Thus the Michaelis constants of 3-O-[14C] methylglucose entry and exit, Kso and Ksi, were 8 and 7 mM, respectively. The equilibrium exchange constant, Bs, and the counterflow constant, Rs, were 9 and 11 mM, respectively. The activity constant for 3-O-methylglucose transport, Fs, defined as V/Km, was 4 ml/h per g. This set of kinetic constants was compatible with a symmetrical mobile-carrier model. In contrast, the Michaelis constant for glucose entry, Kgo, was 2 mM and less than the counterflow constant, Rg (8 mM). This result could be accounted for by slower movement of the glucose-carrier complex than the free carrier. The activity constant for glucose transport, Fg, was 5 ml/h perg.Under aerobic conditions, two of the dissociation-like constants (Ksi and Bs) for 3-O-methylglucose transport were significantly larger than those obtained in anaerobic cells, but the remaining two (Kso and Rs) remained unchanged. The values, for Kso, Ksi, Bs and Rs were 8, 26, 20 and 8 mM, respectively. The activity constant, Fs, decreased to 2 ml/h per g. These changes in kinetic constants were consistent with the hypothesis that anoxia accelerated sugar transport by releasing free carrier that was previously sequestered on the inside of the cell membrane.  相似文献   

3.
The uptake of α-methyl-d-glucoside was stimulated in slices of rat kidney cortex by pretreatment with reduced glutathione. Diamide, an oxidizing agent with high specificity for GSH, caused an inhibition of α-methyl-d-glucoside uptake. These effects appeared to be related specifically to GSH, since dithiothreitol and mercaptoethanol did not increase α-methyl-d-glucoside uptake, and were not as effective as GSH in reversing the effects of diamide. GSH and diamide had no effect on the uptake of another sugar analog, 3-O-methylglucose, which is not actively transported. Kinetic studies indicated that GSH increased the apparent V without affecting Km. The results are discussed in terms of the possible role of GSH in the process of sugar transport.  相似文献   

4.
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6.
The transport of 3-O-methylglucose in white fat cells was measured under equilibrium exchange conditions at 3-O-methylglucose concentrations up to 50 mM with a previously described method (Vinten, J., Gliemann, J. and Østerlind, K. (1976) J. Biol. Chem. 251, 794–800). Under these conditions the main part of the transport was inhibitable by cytochalasin B. The inhibition was found to be of competitive type with an inhibition constant of about 2.5 · 10?7 M, both in the absence and in the presence of insulin (1μM). The cytochalasin B-insensitive part of the 3-O-methylglucose permeability was about 2 · 10?9 cm · s?1, and was not affected by insulin. As calculated from the maximum transport capacity, the half saturation constant and the volume/ surface ratio, the maximum permeability of the fat cell membrane to 3-O-methylglucose at 37°C and in the presence of insulin was 4.3 · 10?6 cm · s?1. From the temperature dependence of the maximum transport capacity in the interval 18–37°C and in the presence of insulin, an Arrhenius activation energy of 14.8 ± 0.44 kcal/mol was found. The corresponding value was 13.9 ± 0.89 in the absence of insulin. The half saturating concentration of 3-O-methylglucose was about 6 mM in the temperature interval used, and it was not affected by insulin, although this hormone increased the maximum transport capacity about ten-fold to 1.7 mmol · s?1 per 1 intracellular water at 37°C.  相似文献   

7.
Band 3 protein, extracted from human erythrocyte membranes by Triton X-100, was recombined with egg lecithin/cholesterol mixtures to form small unilamellar vesicles at a yield of 15–20%. These systems exhibited sulfate fluxes which were inhibitable by stilbene disulfonates and other inhibitors. Maximal inhibition could only be obtained when inhibitors were present at both membrane surfaces. Inhibitor constants I50 were higher than in the native membrane. Quantitatively, transport function was retained at least 60%, as related to the amount of protein involved. Sulfate transport in the recombinates resembled transport in the native membrane with respect to temperature dependence (Ea = 29?32 kcal/mol), pH dependence between pH 6.5 and 7.8, and the relationship between net and exchange fluxes. In contrast to the native cell, concentration dependence was linear up to 80 mM sulfate, which may be indicative of a lowered affinity for the substrate. Lactate transport in these systems, although substantial, was insensitive to stilbene disulfonates as well as to mercurials, indicating that band 3 is not involved in the specific monocarboxylate transfer in the erythrocyte. Anion transport in band 3-lipid recombinates was insensitive to cholesterol between 0 and 27 mol%. Treatment with proteases, while not affecting transport per se, abolished sensitivity to stilbene disulfonate inhibitors. These observations indicate a number of disturbances of band 3 after recombination, in spite of a preservation of the major transport properties.  相似文献   

8.
9.
Unidirectional fluxes of [14C]lactose by whole cells of Escherichia coli under highly energized and partially de-energized (in the presence of CN?) conditions are analyzed kinetically.When the cells are energized, the value for V influx is 0.45 ± 0.01 mM internal concentration increment/s and Kt is 0.26 ± 0.03 mM. At an external concentration of 0.61 mM the steady-state internal concentration is 0.25 M, reached after about 1h. The maximum steady-state concentration ratio is 2 · 103.The efflux process under these conditions is non-saturable, being linearly dependent upon internal concentration over the range 25–250 mM with a first-order rate constant of 8.8 ± 0.2 · 10?4 s?1.The transport in the presence of CN? is active, with a maximum concentration ratio (internal concentration/external concentration) of 104, and the uptake is mimicked by anoxia (< 70 ppm O2).The effects of CN? are to lower the V for influx and to change the efflux from a non-saturable to a saturable process with a value for Kt (60 mM) intermediate between that for energized efflux (> 250 mM) and influxe (0.3–0.6 mM), the latter value not changing appreciably. Partial de-energization thus affects both the influx and efflux processes.  相似文献   

10.
Influx and efflux of glycine have been examined as a function of external and internal Na+ concentrations, respectively, when ΔμNa = 0. With ΔμNa = 0 it was found that at comparable external and cellular Na+ levels, the Km for efflux was larger by an order of magnitude than the value for influx and the V for efflux was several times greater than the V for influx. For both fluxes the major effect of Na+ was to decrease the Km value. The observations are consistent with the conclusion that the Na+-dependent transport system is asymmetric per se. Influx and efflux of glycine were increased in a near linear manner by increasing the Na+ concentration from 13 to 100 mM, the half-time for glycine equilibration being a function of the Na+ concentration in absence of an electrochemical potential difference for Na+. In Na+-free media ([Na+] < 5 mM) equilibration of glycine between cells and medium was not achieved after 60 min at 25°C. With ΔμNa= 0, efflux (or uptake) of glycine was not affected by internal (or external) K+ between 20 and 120 mM suggesting that K+ plays no direct role in Na+-dependent transport of glycine in Ehrlich cells.  相似文献   

11.
10?5 M cyclic AMP has high permeability in human erythrocyte ghosts (p = 0.061 · 10?6cm · s?1). Saturation of influx and efflux occurs. Kztoi = 4.43 mM. Vztoi = 259.6 μM · min?1. Kztio = 0.475 μM. Vztio = 28.3 μM · min?1 at 30°C. Equilibrium exchange entry of cyclic AMP has similar kinetics to zero trans influx, though the system does show counterflow. Cythochalasin B is an apparent competitive inhibitor of cyclic AMP exit. (Ki = 3.9 · 10?7M).Control experiments indicated that cyclic AMP remains intact during incubation with red blood cell ghosts and is contained within the intravesicular space during the transport experiments.  相似文献   

12.
(1) Vanadate (VO3?) fully inhibits the ATP-dependent uncoupled Ca efflux (Ca pump) in dialyzed squid axons. (2) Vanadate inhibits with high affinity. The mean apparent affinity (K12) obtained was 7 μM. (3) Inhibition by vanadate is dependent on Cao. External Ca lead to a release of the inhibitory effect. (K12 ≈ 3 mM). This antagonic effect can be reverted by increasing the vanadate concentration. Internal K+ increases the affinity of the intracellular vanadate binding site. External K+ has no effect on the inhibition. (4) Vanadate has no effect on the Nao-dependent Ca efflux component (forward Na-Ca exchange) in the absence of ATP. In axons containing ATP vanadate modified this component.  相似文献   

13.
Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte (I50 = 6·10?7M). The concentration dependence of the binding to ghosts reveals two saturable components. [14C]Flufenamate binds with high affinity (Kd1 = 1.2·10?7M) to 8.5·105 sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity (Kd2 = 10?4M) to a second set of sites (4.6·107 per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [14C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [14C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [14C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [14C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.  相似文献   

14.
Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K+ concentration (Ki: 200–300 mM) which greatly exceeds that of the growth medium, and a low Na+ concentration (Nai+: 20 mM). Unlike Nai+, Ki+ varies with cell aging.The K+ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K+ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, Ki+ decreases and is partially compensated by a gain in Na+. This substitution completely reverses when metabolic substrate is added (K+ reaccumulation process). Kinetic analysis of K+ movement in cells with steady K+ level shows that most of K+ influx is mediated by an autologous K+-K+ exchange mechanism. On the other hand, during K+ reaccumulation by K+-depleted cells, a different mechanism (a K+ uptake mechanism) with higher transport capacity and affinity drives the net K+ influx. Both mechanisms are energy-dependent.Ouabain and anoxia have no effect on K+ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg2+-dependent ATPase activity.  相似文献   

15.
Glucose transport in human erythrocytes is characterized by a marked asymmetry in the V and Km values for entry and for exit. In addition, they show a high Km and a high V for equilibrium exchange but low Km values for infinite cis and for infinite trans exit and entry. An allosteric pore model has been proposed to account for these characteristics. In this model, substrate-induced conformational changes destabilize the interfaces between protein subunits (the pore gates).Pores doubly occupied from inside destabilize the transport gates and result in high Km and high V transport parameters. This effect is less marked when pores are doubly occupied from outside and therefore transport asymmetry results.  相似文献   

16.
Phosphate transporter of bovine heart mitochondria was purified by solubilization of submitochondrial particles with octylglucoside and fractionation of the extract with ammonium sulfate. After reconstitution into liposomes the purified protein catalyzed phosphate transport which was sensitive to mersalyl and other SH reagents. Transport measured either as PiOH or PiPi exchange was proportional to protein concentration and time. The PiOH but not the PiPi exchange was stimulated several fold by valinomycin plus nigericin in the presence of K+. The reconstituted system provides a suitable assay during purification of the mitochondrial phosphate transporter.  相似文献   

17.
The mechanism of mitochondrial oxaloacetate transport has been investigated by measuring the rate and the extent of exchange reactions between intramitochondrial anions and added oxaloacetate. The exchange between oxaloacetate and intramitochondrial oxoglutarate is insensitive to mersalyl at a concentration which completely inhibits the dicarboxylate carrier. Oxaloacetate causes efflux of intramitochondrial Pi, malonate, and malate. Mersalyl inhibits completely the oxaloacetate/Pi exchange, but only partially the oxaloacetate/malonate and the oxaloacetate/malate exchanges. The inhibition of the last two reactions decreases on increasing the time of incubation. Butylmalonate inhibits more than phenylsuccinate the exchange oxaloacetateout/32Piin, whereas phenylsuccinate is a more effective inhibitor than butylmalonate of the oxaloacetateout/[14C]oxoglutaratein exchange. The apparent Km values ranged from 0.6 to 1.2 mm for the oxaloacetate/oxoglutarate exchange and from 6.5 to 10 mm for the oxaloacetate/Pi exchange. The inhibition of oxoglutarate uptake by oxaloacetate is competitive. Oxaloacetate inhibits the malonate/Pi exchange competitively and it is a noncompetitive inhibitor of the PiPi exchange. It is concluded that oxaloacetate may be transported across the mitochondrial membrane by the oxoglutarate carrier and, much less effectively, by the dicarboxylate carrier. The implications of these findings are discussed.  相似文献   

18.
The proton efflux from intact, anaerobic Escherichiacoli cells following a small oxygen pulse is both slow (t1M2~-10s) and inefficient (H+O~-0.5. Very low levels (<80 nM) of the proton ionophore carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), which have no detectable effect upon active transport, cause a 3–5 fold stimulation in the extent of proton efflux without affecting the efflux rate. At slightly higher concentrations of FCCP (80 nM to 0.5 μM), a sharp inhibition of this increased proton efflux occurs, with the H+O ratio obtained in the presence of 0.5 μM FCCP approximately equal to that obtained in the absence of FCCP. Still higher concentrations of FCCP (> 1 μM), which inhibit active transport, cause a further gradual decrease in the H+O ratio. The unusual increase in the apparent efficiency of H+ efflux by <80 nM FCCP is not accompanied by an increase in the rate of membrane deenergization following an O2 pulse, although such an increase is seen with the higher (uncoupling) FCCP concentrations.  相似文献   

19.
The uptake of [32P]phosphate into human red blood cells was inhibited (Ki = 0.6 mM) by the sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). 2-Nitro-5-thiobenzoic acid (NTB), the reduced form of DTNB, was a less potent inhibitor (Ki = 7 mM). The inhibition of anion transport by DTNB could be reversed by washing DTNB-treated cells with isotonic buffer, or by incubating DTNB-treated cells with 2-mercaptoethanol, which converted DTNB to NTB. DTNB competitively inhibited the binding of 4-[14C]-benzamido-4′-aminostilbene-2,2′-disulfonate, a potent inhibitor of anion transport (Ki = 1?2 μM), to band 3 protein in cells and ghost membranes. These results suggest that the stilbene-disulfonate binding site in band 3 protein can readily accommodate the organic anion DTNB, and that inhibition by DTNB was not due to reaction with an essential sulfhydryl group.  相似文献   

20.
(1) The effects of vanadate of hexose transport, 45Ca-exchange and (Na+, K+)-contents have been characterized in isolated adipose tissue and skeletal muscles of the rat. (2) In whole epididymal fat pads, vanadate (0.5–5.0 mM) markedly stimulated the uptake of 2-deoxyl[14C]glucose as well as the efflux of 3-O-[14C]methylglucose. (3) Within the same concentration range, vanadate induced an early increase in 45Ca-washout from preloaded fat pads. The maximum increases in the fractional losses of 3-O-[14C]methylglucose and 45Ca were significantly correlated (P < 0.001, r = 0.98). (4) In extensor digitorum longus and soleus muscles, vanadate (0.5–5.0 mM) stimulated the efflux of 3-O-[14C]methylglucose and this effect was preceded by a rise in the washout of 45Ca. The maximum increases in the fractional losses of 3-O-[14C]methyglucose and 45Ca were significantly correlated (P < 0.005, r = 0.98). (5) In extensor digitorum longus and soleus muscles, vanadate increased K+-contents and decreased Na+ contents. (6) The stimulation of 45Ca-washout presumably reflects an increase in the cytoplasmic Ca2+ level, brought about by an inhibitory effect of vanadate on the Ca2+-sensitive ATPase of the sarcoplasmic or the endoplasmic reticulum. As demonstrated for most other insulin-like agents (Sørensen, S.S., Christensen, F. and Clausen, T. (1980) Biochim. Biophys. Acta 602, 433–445), the stimulating effect of vanadate on glucose transport appears to be associated with or mediated by a rise in the cytoplasmic Ca2+ level.  相似文献   

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