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1.
D Khananshvili 《Biochemistry》1990,29(10):2437-2442
In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.  相似文献   

2.
Na-Ca exchange current was measured at various concentrations of internal Na [( Na]i) and Ca [( Ca]i) using intracellular perfusion technique and whole-cell voltage clamp in single cardiac ventricular cells of guinea pig. Internal Ca has an activating effect on Nai-Cao exchange beginning at approximately 10 nM and saturating at approximately 50 nM with a half maximum [Ca]i (Km[Ca]i) of 22 nM (Hill coefficient, 3.7). Measurement of Nai-Cao exchange current at various concentration of [Na]i revealed an apparent Km[Na]i of 20.7 +/- 6.9 mM (n = 14) with imax of 3.5 +/- 1.2 microA/microF. For [Ca]i transported by the exchange, a Km[Ca]i of 0.60 +/- 0.24 microM (n = 8) with an imax of 3.0 +/- 0.54 microA/microF was obtained by measuring Nao-Cai exchange current. These values are apparently different from the values for the external binding site which have been reported previously. Whether Na and Ca compete for the external binding site, and if so, how it affects the binding constants was then investigated. Outward Nai-Cao exchange current became larger by reducing [Na]o. The double reciprocal plot of the current magnitude and [Ca]o at different [Na]o revealed a competitive interaction between Na and Ca. In the absence of competitor [Na]o, an apparent Km[Ca]o of 0.14 mM was obtained. When comparing internal and external Km values, the external value is markedly larger than the internal one and thus we conclude that binding sites of the Na-Ca exchange molecule are at least apparently asymmetrical between the inside and outside of the membrane.  相似文献   

3.
The transport of Na+ and Ca2+ ions in the cardiac Na(+)-Ca2+ exchanger can be described as separate events (Khananshvili, D. (1990) Biochemistry 29, 2437-2442). Thus, the Na(+)-Na+ and Ca(2+)-Ca2+ exchange reactions reflect reversible partial reactions of the transport cycle. The effect of diffusion potentials (K(+)-valinomycin) on different modes of the Na(+)-Ca2+ exchanger (Na(+)-Ca2+, Ca(2+)-Ca2+, and Na(+)-Na+ exchanges) were tested in reconstituted proteoliposomes, obtained from the Triton X-100 extracts of the cardiac sarcolemmal membranes. The initial rates of the Nai-dependent 45Ca-uptake (t = 1 s) were measured in EGTA-entrapped proteoliposomes at different voltages. At the fixed values of voltage [45 Ca]o was varied from 4 to 122 microM, and [Na]i was saturating (150 mM). Upon varying delta psi from -94 to +91 mV, the Vmax values were increased from 9.5 +/- 0.5 to 26.5 +/- 1.5 nmol.mg-1.s-1 and the Km from 17.8 +/- 2.5 to 39.1 +/- 5.2 microM, while the Vmax/Km values ranged from only 0.53 +/- 0.08 to 0.73 +/- 0.17 nmol.mg-1.s-1.microM-1. The equilibrium Ca(2+)-Ca2+ exchange was voltage sensitive at very low [Ca]o = [Ca]i = 2 microM, while at saturating [Ca]o = [Ca]i = 200 microM the Ca(2+)-Ca2+ exchange became voltage-insensitive. The rates of the equilibrium Na(+)-Na+ exchange appears to be voltage insensitive at saturating [Na]o = [Na]i = 160 mM. Under the saturating ionic conditions, the rates of the Na(+)-Na+ exchange were at least 2-3-fold slower than the Ca(2+)-Ca2+ exchange. The following conclusions can be drawn. (a) The near constancy of the Vmax/Km for Na(+)-Ca2+ exchange at different voltages is compatible with the ping-pong model proposed previously. (b) The effects of voltage on Vmax of Na(+)-Ca2+ exchange are consistent with the existence of a single charge carrying transport step. (c) It is not yet possible to clearly assign this step to the Na+ or Ca2+ transport half of the cycle although it is more likely that 3Na(+)-transport is a charge carrying step. Thus, the unloaded ion-binding domain contains either -2 or -3 charges (presumably carboxyl groups). (d) The binding of Na+ and Ca2+ appears to be weakly voltage-sensitive. The Ca(2+)-binding site may form a small ion-well (less than 2-3 A).  相似文献   

4.
1. The fluorescent intensity of the dye 3,3'-dipropylthiodicarbocyanine iodide was measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potentials under a variety of different ionic and metabolic conditions. 2. In the presence of valinomycin, fluorescent intensity is dependent on log [K+]medium (the fluorescent intensity increased with increasing [K+]medium) where K+ replaced Na+ in the medium. Cellular K+ content also influenced fluorescent intensity in the presence of valinomycin. With lower cellular K+, fluorescent intensity in the presence of valinomycin for any given concentration was increased. 3. In the presence of gramicidin fluorescent intensity was highest in Krebs-Ringer and decreased with the substitution of choline+ for Na+. 4. The observations with ionophores are consistent with the hypothesis that the dye monitors membrane potential in these cells with an increase in fluorescence indicating membrane depolarization (internal becomes more positive). 5. The estimated membrane potentials were influenced by the way in which the cells were treated. Upon dilution of the cells from 1 in 20 to 1 in 300 the initial estimations were between -50 and -60 mV. With incubation at 1 in 300 dilution for 1 h at room temperature or a 37 degrees C, the membrane potentials ranged from -18 to -42 mV. 6. Estimations of membrane potential on the basis of chloride distribution (Cl-cell/Cl-medium) in equilibrated cells ranged from -13 to -32 mV. 7. Addition of glucose to cells equilibrated at 37 degrees C for 30 min in the presence of rotenone led to a decrease in fluorescent intensity indicating hyperpolarization. Addition of ouabain in turn led to a 70 to 100% reversal of fluorescent intensity. This hyperpolarization is therefore probably due to the electrogenic activity of the sodium pump. 8. The addition of amino acids known to require external Na+ for transport increased fluorescent intensity (depolarization) reaching a maximum at higher concentrations of amino acids. Plots of 1/deltafluorescence vs. 1/[glycine] were linear with an apparent Km of 2-3 mM. The increase in fluorescence with amino acids always required external Na+. Plots of 1/fluorescence vs. 1/[Na+]medium were also linear with an apparent Km of 29 mM. These apparent Km values compare favorably with those derived from amino acid transport studies using tracers. These data indicate that the Na+-dependent transport of amino acids in these cells is electrogenic.  相似文献   

5.
Specific effects of spermine on Na+,K+-adenosine triphosphatase   总被引:2,自引:0,他引:2  
Specific effects of spermine on Na+,K+-ATPase were observed using an enzyme partially purified from rabbit kidney microsomes by extraction with deoxycholate. 1. Spermine competed with K+ for K+-dependent, ouabain-sensitive nitrophenylphosphatase. The K1 for spermine was 0.075 mm in the presence of 1 mM Mg2+ and 5 mM p-nitrophenylphosphate at pH 7.5. 2. spermine activated Na+,K+-ATPase over limited concentration ranges of K+ and Na+ in the presence of 0.05 mM ATP. The spermine concentration required for half maximal activation was 0.055 mM in the presence of 1 mM K+, 10 mM Na+, 1 mM Mg2+, and 0.05 mM ATP. 3. The activation of Na+,K4-ATPase was not due to substitution of spermine for K+, Na+, or Mg2+. 4. When the concentration of K+ or Na+ was extremely low, or in excess, spermine did not activate Na+,K+-ATPase, but inhibited it slightly. 5. Plots of 1/v vs. 1/[ATP] at various concentrations of spermine showed that spermine decreased the Km for ATP without changing the Vmax. 6. Plots of 1/v vs. 1/[ATP] at concentrations of K+ from 0.05 mM to 0.5 mM showed that K+ increased the Km for ATP with increase in the Vmax in the presence of 0.2 mM spermine similarly to that in the absence of spermine. The contradictory effects of spermine on this enzyme system suggest that the K+-dependent monophosphatase activity does not reflect the second half (the dephosphorylation step) of the Na+,K+-ATPase catalytic cycle.  相似文献   

6.
Isolated rat brain synaptosomes accumulated L-asparagine with a Km value of 348 microM and a Vmax value of 3.7 nmol/mg of protein/min at 28 degrees C. Uptake of L-asparagine was inhibited by the presence of L-glutamine, whereas transport of L-glutamine was blocked by L-asparagine. Alanine, serine, cysteine, threonine, and, in particular, leucine were also inhibitory whereas alpha-(methylamino)isobutyrate, ornithine, lysine, arginine, and glutamate were much less effective blockers. Transport of L-asparagine had a substantial sodium-dependent component, whereas that of the D-stereoisomer was almost unaffected by the presence or absence of the cation. L-Asparagine was accumulated to a maximal gradient, [L-Asn]i/[L-Asn]o, of 20-30, and this value was reduced to 5-6 by withdrawal of sodium or addition of high [KCI]. A plot of log [Na+]o/[Na+]i against the log [L-Asn]i/[L-Asn]o had a slope close to I, which indicates that a single sodium ion is transported inward with each asparagine molecule. It is postulated that uptake of L-asparagine occurs, to a large extent, in cotransport with Na+ and that it utilizes the sodium chemical gradient and the membrane electrical potential as the source of energy. The similarity between the L-asparagine and L-glutamine transport systems and the reciprocal inhibition of influx of the two amino acids suggest that the same mechanism is responsible for glutamine accumulation. This could explain the high [Gln]i maintained by the brain in vivo.  相似文献   

7.
1. The pH-dependence is considered of a reaction between E and S that proceeds through an intermediate ES under "Briggs-Haldane' conditions, i.e. there is a steady state in ES and [S]o greater than [E]T, where [S]o is the initial concentration of S and [E]T is the total concentration of all forms of E. Reactants and intermediates are assumed to interconvert in three protonic states (E equilibrium ES; EH equilibrium EHS; EH2 equilibrium EH2S), but only EHS provides products by an irreversible reaction whose rate constant is kcat. Protonations are assumed to be so fast that they are all at equilibrium. 2. The rate equation for this model is shown to be v = d[P]/dt = (kcat.[E]T[S]o/A)/[(KmBC/DA) + [S]o], where Km is the usual assembly of rate constants around EHS and A-D are functions of the form (1 + [H]/K1 + K2/[H]), in which K1 and K2 are: in A, the molecular ionization constants of ES; in B, the analogous constants of E; in C and D, apparent ionization constants composed of molecular ionization constants (of E or ES) and assemblies of rate constants. 3. As in earlier treatments of this type of reaction which involve either the assumption that the reactants and intermediate are in equilibrium or the assumption of Peller & Alberty [(1959) J. Am. Chem. Soc. 81, 5907-5914] that only EH and EHS interconvert directly, the pH-dependence of kcat. is determined only by A. 4. The pH-dependence of Km is determined in general by B-C/A-D, but when reactants and intermediate are in equilibrium, C identical to D and this expression simplifies to B/A. 5. The pH-dependence of kcat./Km, i.e. of the rate when [S]o less than Km, is not necessarily a simple bell-shaped curve characterized only by the ionization constants of B, but is a complex curve characterized by D/B-C. 6. Various situations are discussed in which the pH-dependence of kcat./Km is determined by assemblies simpler than D/B-C. The special situation in which a kcat./Km-pH profile provides the molecular pKa values of the intermediate ES complex is delineated.  相似文献   

8.
The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.  相似文献   

9.
Amino acid transport in horse erythrocytes is regulated by three co-dominant allelomorphic genes coding for high-affinity transport activity (system asc1), low-affinity transport activity (system asc2) and transport-deficiency, respectively. The asc systems are selective for neutral amino acids of intermediate size, but unlike conventional system ASC, do not require Na+ for activity. In the present series of experiments we have used a combined kinetic and genetic approach to establish that dibasic amino acids are also asc substrates, systems asc1 and asc2 representing the only mediated routes of cationic amino acid transport in horse erythrocytes. Both transporters were found to exhibit a strong preference for dibasic amino acids compared with neutral amino acids of similar size. Apparent Km values (mM) for influx via system asc1 were L-lysine (9), L-ornithine (27), L-arginine (27), L-alanine (0.35). Corresponding Vmax estimates (mmol/l cells per h, 37 degrees C) were L-lysine (1.65), L-ornithine (2.15), L-arginine (0.54), L-alanine (1.69). Apparent Km values for L-lysine and L-ornithine influx via system asc2 were approximately 90 and greater than 100 mM, respectively, with Vmax values greater than 2 and greater than 1 mmol/l cells per h, respectively. Apparent Km and Vmax values for L-alanine uptake by system asc2 were 14 mM and 6.90 mmol/l cells per h. In contrast, L-arginine was transported by system asc2 with the same apparent Km as L-alanine (14 mM), but with a 77-fold lower Vmax. This dibasic amino acid was shown to cause cis- and trans-inhibition of system asc2 in a manner analogous to its interaction with system ASC, where the side-chain guanidinium group is considered to occupy the Na+-binding site on the transporter. Concentrations of extracellular L-arginine causing 50% inhibition of zero-trans L-alanine influx and half-maximum inhibition of L-alanine zero-trans efflux were 14 mM (extracellular L-alanine concentration 15 mM) and 3 mM (intracellular L-alanine concentration 15.5 mM), respectively. We interpret these observations as evidence of structural homology between the horse erythrocyte asc transporters and system ASC. Physiologically, intracellular L-arginine may function as an endogenous inhibitor of system asc2 activity.  相似文献   

10.
Intracellular pH (pHi) in squid giant axons recovers from acid loads by means of a Na(+)-dependent Cl-HCO3 exchanger, the actual mechanism of which might be exchange of: (i) external Na+ and HCO3- for internal Cl- and H+, (ii) Na+ plus two HCO3- for Cl-, (iii) Na+ and CO3= for Cl-, or (iv) the NaCO3- ion pair for Cl-. Here we examine sensitivity of transport to changes of extracellular pH (pHo) in the range 7.1-8.6. We altered pHo in four ways, using: (i) classical "metabolic" disturbances in which we varied [HCO3-]o, [NaCO3-]o, and [CO3=]o at a fixed [CO2]o; (ii) classical "respiratory" disturbances in which we varied [CO2]o, [NaCO3-]o, and [CO3=]o at a fixed [HCO3-]o; (iii) novel mixed-type acid-base disturbances in which we varied [HCO3-]o and [CO2]o at a fixed [CO3=]o and [NaCO3-]o; and (iv) a second series of novel mixed-type disturbances in which we varied [CO2]o, [CO3=]o, and [Na+]o at a fixed [HCO3-]o and [NaCO3-]o. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. The equivalent acid extrusion rate (JH) was computed from the rate of pHi recovery (i.e., increase) in the presence of Na+ and HCO3-. When pHo was varied by method (i), which produced the greatest range of [CO3=]o and [NaCO3-]o values, JH increased with pHo in a sigmoidal fashion; the relation was fitted by a pH titration curve with a pK of approximately 7.7 and a Hill coefficient of approximately 3.0. With method (ii), which produced smaller changes in [CO3=]o and [NaCO3-]o, JH also increased with pHo, though less steeply. With method (iii), which involved changes in neither [CO3=]o nor [NaCO3-]o, JH was insensitive to pHo changes. Finally, with method (iv), which involved changes in neither [HCO3-] nor [NaCO3-]o, but reciprocal changes in [CO3=]o and [Na+]o, JH also was insensitive to pHo changes. We found that decreasing pHo from 8.6 to 7.1 caused the apparent Km for external HCO3- ([Na+]o = 425 mM) to increase from 1.0 to 26.7 mM, whereas Jmax was relatively stable. Decreasing pHo from 8.6 to 7.4 caused the apparent Km values for external Na+ ([HCO3-]o = 48 mM) to increase from 8.6 to 81 mM, whereas Jmax was relatively stable.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

11.
Uptake and inhibitory kinetics of [3H]L-threonine were evaluated in preparations of pig jejunal brush border membrane vesicles. Uptake of [3H]L-threonine under O-trans, Na+ gradient, and O-trans, Na(+)-free conditions was best described by high affinity transport (Km < 0.01 mM) plus a nonsaturable component. The maximal velocity of transport was 3-fold greater under Na+ gradient conditions. 100 mM concentrations of all of the dipolar amino acids and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid caused complete inhibition of [3H]L-threonine transport under Na+ gradient and Na(+)-free conditions. Imino acids, anionic amino acids, cationic amino acids, and methylamino-isobutyric acid caused significant partial inhibition of L-threonine uptake. Inhibitor concentration profiles for proline and lysine were consistent with low affinity competitive inhibition. The Ki values of alanine and phenylalanine approximated 0.2 and 0.5 mM, respectively, under both Na+ gradient and Na(+)-free conditions. These data indicate that the transport system available for L-threonine in the intestinal brush border membrane (system B) is functionally distinct from other amino acid transport systems. Comparison of kinetics parameters in the presence and absence of a Na+ gradient suggests that both partially and fully loaded forms of the carrier can function to translocate substrate and that Na+ serves to accelerate L-threonine transport by a mechanism that does not involve enhanced substrate binding.  相似文献   

12.
Uptake of L-2,4-diaminobutyric acid (DABA), a positively charged analogue of gamma-aminobutyric acid (GABA), by a synaptosomal fraction isolated from rat brain occurred with a Km of 54 +/- 12 microM and a Vmax of 1.3 +/- 0.2 nmol/min/mg protein. The transport of DABA was inhibited competitively by GABA whereas that of GABA was affected in the same manner by addition of DABA. The maximal accumulation of DABA ([DABA]i/[DABA]c) was observed to increase as the second power of the transmembrane electrical potential ([K+]i/[K+]e) and the first power of the sodium ion concentration gradient. These findings indicate that DABA is transported on the GABA carrier with a net charge of +2, where one charge is provided by the cotransported Na+ and the second is contributed by the amino acid itself. Since uptake of GABA, an electroneutral molecule, is accompanied by transfer of two sodium ions, the results obtained with DABA suggest that one of the sodium binding sites on the GABA transporter is in proximity to the amino acid binding site.  相似文献   

13.
The intracellular pH-regulating mechanism of the squid axon was examined for its dependence on the concentrations of external Na+ and HCO3-, always at an external pH (pHo) of 8.0. Axons having an initial intracellular pH (pHi) of approximately 7.4 were internally dialyzed with a solution of pH 6.5 that contained 400 mM Cl- and no Na+. After pHi had fallen to approximately 6.6, dialysis was halted, thereby returning control of pHi to the axon. With external Na+ and HCO-3 present, intracellular pH (pHi) increased because of the activity of the pHi-regulating system. The acid extrusion rate (i.e., equivalent efflux of H+, JH) is the product of the pHi recovery rate, intracellular buffering power, and the volume-to-surface ratio. The [HCO3-]o dependence of JH was examined at three fixed levels of [Na+]o: 425, 212, and 106 mM. In all three cases, the apparent Jmax was approximately 19 pmol X cm-2 X s-1. However, the apparent Km (HCO3-) was approximately inversely proportional to [Na+]o, rising from 2.6 to 5.4 to 9.7 mM as [Na+]o was lowered from 425 to 212 to 106 mM, respectively. The [Na+]o dependence of JH was similarly examined at three fixed levels of [HCO3-]o: 12, 6, and 3 mM. The Jmax values did not vary significantly from those in the first series of experiments. The apparent Km (Na+), however, was approximately inversely related to [HCO3-]o, rising from 71 to 174 to 261 mM as [HCO3-]o was lowered from 12 to 6 to 3 mM, respectively. These results agree with the predictions of the ion-pair model of acid extrusion, which has external Na+ and CO3= combining to form the ion pair NaCO3-, which then exchanges for internal Cl-. When the JH data are replotted as a function of [NaCO3-]o, data from all six groups of experiments fall along the same Michaelis-Menten curve, with an apparent Km (NaCO3-) of 80 microM. The ordered and random binding of Na+ and CO3= cannot be ruled out as possible models, but are restricted in allowable combinations of rate constants.  相似文献   

14.
Characterization of the ascorbic acid transport by 3T6 fibroblasts   总被引:2,自引:0,他引:2  
Ascorbic acid transport by 3T6 mouse skin fibroblasts has been characterized using radiometric technique with L-[1-14C]ascorbic acid under the conditions in which oxidation of ascorbic acid was prevented by addition of 1 mM thiourea. The ascorbate transport is temperature-dependent with the energy of activation E and Q10 of 13.3 kcal/mol and 2.0, respectively. The transport requires energy and exhibits Michaelis-Menten kinetics with an apparent Km of 112 microM and Vmax of 158 pmol/min per mg protein, when the extracellular Na+ concentration is 150 mM. The ascorbate transport requires presence of extracellular Na+ and can be inhibited by ouabain treatment. At 40 and 200 microM ascorbate concentrations, respectively, 1.4 and 1.0 moles of Na+ bound the transporter molecule per each mole of ascorbate transported. Increased Na+ binding to the transporter at lower ascorbate concentration may signify multiple Na+-binding sites or ascorbate concentration dependent conformational changes in the transporter molecule. Increasing Na+ concentration decreases Km without affecting Vmax, suggesting that Na+ increases affinity of ascorbate for the transporter molecule without affecting translocation process. An increase in ascorbate concentration reduces the number of Na+ bound to the transporter from 1.4 to 1.0. The ascorbate transport is stimulated by Ca2+ and other divalent cations. The mechanism of stimulation by Ca2+ is not clear. Calcium increases both the Km and Vmax. The data presented support the hypothesis that the ascorbate transport by 3T6 fibroblasts is an energy and temperature-dependent active process driven by the Na+ electrochemical gradient. A potent inhibitor of ascorbate transport is also demonstrated in human serum.  相似文献   

15.
For bovine erythrocyte acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7), the Michaelis parameters Vmax., and Km for the natural substrate acetylcholine were estimated as a function of pH and sodium chloride concentration by the pH-stat method. A single dissociation constant for Na+ binding (K = 7 X 10(-3) M) suffices to explain the salt dependence of Vmax./Km and of Km as well as the pH dependence of Vmax./Km and Vmax., Km being pH independent. This finding provides evidence for a specific effect of Na+, presumably by binding at the anionic subsite of the active centre. Na+ binding causes a 50-fold decrease in kcat./Km as well as a decrease of one unit in the pKa of both kcat./Km and kcat.. The intrinsic pKa in the absence of salt at 25 degrees C is about 7.5. Comparison of the degree of fit of the data to the Debeye-Huckel equation, in accordance with an alternative general salt effect, as well as published data for sodium and potassium chlorides also favour a specific salt effect.  相似文献   

16.
Microelectrode measurements of apical membrane potentials (Va) in absorptive cells of isolated Necturus intestine showed that, in the presence or absence of external Na+, 10 mM lysine added to the mucosal medium caused rapid depolarization followed by slower repolarization of Va. In Na+-free media the effects of 10 mM lysine on Va were abolished by 10 mM leucine which alone had no effect on Va under these conditions. This indicates that uncoupled electrodiffusion of lysine plays little or no role in lysine entry across the brush-border membrane. When external Na+ was greater than 10 mM the maximum depolarization of Va (delta Va') induced by [Lys] ranging from 5 to 30 mM was a simple saturable function of [Lys]. In Na+-free media, the relationship between delta Va' and [Lys] was biphasic. At first, delta Va' increased with increasing [Lys] reaching a maximum at 10 mM lysine. When [Lys] was further increased, delta Va' declined progressively to reach zero or near zero values. A single transport pathway model is proposed to account for rheogenic lysine entry across the brush-border membrane in the presence and absence of Na+. This postulates an amino acid transporter in the membrane with two binding sites. One is an amino acid site specific for the alpha-amino-alpha-carboxyl group. The other is a Na+ site. Neutral amino acids (e.g. leucine) compete with lysine for the amino acid site. The Na+ site has some affinity for the epsilon-amino group of lysine. When external Na+ is high the Na+ site is essentially 'saturated' with Na+ and formation of a mobile complex between an amino acid and the transporter depends in a saturable fashion on amino acid concentration. In Na+-free media or in media containing low [Na+]; at low external [Lys] the epsilon-amino group of a lysine molecule (simultaneously attached to the amino acid site) interacts with the Na+ site to form a mobile complex, as external [Lys] is increased, attachment of different lysine molecules to each site of an increasing number of transporters to form nontransported or poorly transported complexes results in substrate inhibition of the rheogenic lysine transport process.  相似文献   

17.
The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed.  相似文献   

18.
The effects of some gangliosides on active uptake of nonmetabolizable alpha-aminoisobutyric acid (AIB) and Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined during aerobic incubation at 37 degrees C for 2 h. In NG, amino acid uptake was greatly accelerated with the addition of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylgluc osyl ceramide (GM1) (85%) and also with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide (GM2) or [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetyl- neuraminyl]-galactosylglucosyl ceramide (GD1a) (43% each) compared with a nonaddition control at a 5 nM concentration. Under identical conditions, Na+, K+-ATPase activity was strongly stimulated with GM1 (180%) and GD1a (93%), whereas Ca2+, Mg2+-ATPase activity showed no change. In SCG, on the other hand, AIB uptake was apparently inhibited (-27%) by addition of GM1, with a slight decrease in Na+, K+-ATPase but no change in Ca2+, Mg2+-ATPase activity in the tissue. Both asialo-GM1, in which N-acetylneuraminic acid is deficient, and Forssman glycolipid, which is not present in nervous tissue, failed to produce any significant increase in both SCG and NG not only in amino acid uptake, but also in Na+, K+-ATPase activity. A kinetic study of active AIB uptake showed that GM1 ganglioside produced an increase in Km with no change in Vmax in SCG, whereas it caused a decrease in Km with a slight increase in Vmax in NG. Treatment of NG and SCG with neuraminidase from Vibrio cholerae, an enzyme that split off sialic acid from polysialoganglioside, leaving GM1 intact, caused little inhibition of the amino acid uptake.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

19.
The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.  相似文献   

20.
Mouse ascites-tumour cells oxidizing lactate, in a modified Ringer solution, concentrated 2-aminoisobutyrate, L-methionine or 2-(methylamino)isobutyrate about 20-fold from a 0.4 mM solution in the presence of 2-3 micrograms of nigericin/mg cellular dry wt. The ionophore increased cellular [Na+] to almost 100 mM when extracellular [Na+] was about 45 mM. Either valinomycin or the two mitochondrial inhibitors oligomycin and antimycin acting together each markedly lowered the extent to which the tumour cells concentrated amino acid, from the above factor of about 20 to roughly 2-fold. Ouabain (1 mM) had a similar effect, and further raised cellular [Na+]. The sodium pump appeared to be closely involved in amino acid uptake under these conditions.  相似文献   

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