Derepression of the high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb+ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the Vmax of the low-affinity system has decreased from about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.     

Cotransport of phosphate and sodium by yeast

Phosphate uptake by yeast at pH 7.2 is mediated by two mechanisms, one of which has a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 30 μM and is independent of sodium, and a sodium-dependent mechanism with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.6 μM, both $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values with respect to monovalent phosphate. The sodium-dependent mechanism has two sites with affinity for Na⁺, with affinity constants of 0.04 and 29 mM. Also lithium enhances phosphate uptake; the affinity constants for lithium are 0.3 and 36 mM. Other alkali ions do not stimulate phosphate uptake at pH 7.2. Rubidium has no effect on the stimulation of phosphate uptake by sodium.Phosphate and arsenate enhance sodium uptake at pH 7.2. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of this stimulation with regard to monovalent orthophosphate is about equal to that of the sodium-dependent phosphate uptake.The properties of the cation binding sites of the phosphate uptake mechanism and those of the phosphate-dependent cation transport mechanism have been compared. The existence of a separate sodium-phosphate cotransport system is proposed.     

Effect of N-ethylmaleimide on leucine transport in the chang liver cell. II. Effect on the kinetics of Na+-independent transport

The Na⁺-independent leucine transport system is resolved into two components by their different affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ about 44 μM and 8.0 mM) for leucine in the Chang liver cell. Treatment of the cells with $\text{N-}\text{ethylmaleimide}$ (1 mM) specifically stimulates the high-affinity component of the Na⁺-independent system by greatly increasing its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value, whereas the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value of the low-affinity component is markedly lowered. The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is reduced by prior treatment of the cells with 2,4-dinitrophenol, but this phenomenon seems to be irrelevant to the ATP-depleting action of the uncoupler. The treatment with 2,4-dinitrophenol has been found not to be inhibitory on the subsequent Na⁺-independent leucine uptake itself. Treatment with dibucaine, a phospholipid-interacting drug, also reduces to varying degrees (depending on its concentration) the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the subsequent leucine uptake, although pretreatment with dibucaine can stimulate the Na⁺-independent leucine uptake itself. We conclude that the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is not correlated with the energy level of cell, but involves the perturbation of the membrane bilayer structures.     

Ligand-dependent tryptic inactivation of the ouabain sensitivity of ADP-ATP exchange catalyzed by canine renal Na+, K+-ATPase.

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 $\text{P}{}_{\mathrm{i}}\text{OH}$ or $\text{P}{}_{\mathrm{i}}\text{P}{}_{\mathrm{i}}$ exchange was proportional to protein concentration and time. The $\text{P}{}_{\mathrm{i}}\text{OH}$ but not the $\text{P}{}_{\mathrm{i}}\text{P}{}_{\mathrm{i}}$ 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.     

A chloride requirement for Na+-dependent amino-acid transport by brush border membrane vesicles isolated from the intestine of a mediterranean teleost (Boops salpa)

The uptake of d-glucose, 2-aminoisobutyric acid and glycine was studied with intestinal brush border membrane vesicles of a marine herbivorous fish: Boops salpa. The uptake of these three substances is stimulated by an Na⁺ electrochemical gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{C}{}_{\mathrm{<mtext>in</mtext>}}$). For glucose, an increase of the electrical membrane potential generated by a concentration gradient of the liposoluble anion, SCN^?, increases the Na⁺-dependent transport. This responsiveness to the membrane potential was confirmed by valinomycin. Differently from glucose, uptake of glycine and 2-aminoisobutyric acid requires, besides the Na⁺ gradient, the presence of Cl^? on the external side of the vesicles. In the absence of Cl^?, amino acid uptake is not stimulated by the Na⁺ gradient and is not influenced by an electrical membrane potential generated by SCN^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) or by a K⁺ diffusion potential ($\text{C}{}_{\mathrm{<mtext>in</mtext>}}\text{>C}{}_{\mathrm{<mtext>out</mtext>}}$). This Cl^? requirement differs from the Na⁺ requirement, since a Cl^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) does not result in an accumulation of glycine or 2-aminoisobutyric acid similar to that produced by an Na⁺ gradient.     

Sucrose transport by Streptococcus mutans. Evidence for multiple transport systems

The transport of sucrose by selected mutant and wild-type cells of Streptococcus mutans was studied using washed cocci harvested at appropriate phases of growth, incubated in the presence of fluoride and appropriately labelled substrates. The rapid sucrose uptake observed cannot be ascribed to possible extracellular formation of hexoses from sucrose and their subsequent transport, formation of intracellular glycogen-like polysaccharide, or binding of sucrose or extracellular glucans to the cocci. Rather, there are at least three discrete transport systems for sucrose, two of which are $\text{phospho}\text{enol}\text{pyruvate-dependent}$ phosphotransferases with relatively low apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values and the other a non-phosphotransferase (non-PTS) third transport system (termed TTS) with a relatively high apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. For strain 6715-13 mutant 33, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values are 6.25·10^?5 M, 2.4·10^?4 M, and 3.0·10^?3 M, respectively; for strain NCTC-10449, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values are 7.1·10^?5 M, 2.5·10^?4 M and 3.3·10^?3 M, respectively. The two lower $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ systems could not be demonstrated in mid-log phase glucose-adapted cocci, a condition known to repress sucrose-specific phosphotransferase activity, but under these conditions the highest $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ system persists. Also, a mutant devoid of sucrose-specific phosphotransferase activity fails to evidence the two high affinity (low apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) systems, but still has the lowest affinity (highest $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$) system. There was essentially no uptake at 4°C indicating these processes are energy dependent. The third transport system, whose nature is unknown, appears to function under conditions of sucrose abundance and rapid growth which are known to repress $\text{phospho}\text{enol}\text{pyruvate-dependent}$ sucrose-specific phosphotransferase activity in S. mutans. These multiple transport systems seem well-adapted to S. mutans which is faced with fluctuating supplies of sucrose in its natural habitat on the surfaces of teeth.     

Energization of sulfate transport in yeast

Sulfate uptake by Saccharomyces cerevisiae is stimulated about 12-fold by preincubation of cells with 1% d-glucose or 1% ethanol. The $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ remains unchanged (0.34–0.38 mM), the $\text{J}{}_{\mathrm{<mtext>mar</mtext>}}$ increase from 18–20 to 195–230 and 170–185 nmol/min per g dry wt., respectively, after glucose and ethanol preincubation. The stimulation involves protein synthesis (it is suppressed by cycloheximide), has a half-time of 18 min and requires mitochondrial respiration (no or low effect in respiration-deficient mutants and those lacking ADP-ATP transport in mitochondria, as well as after anaerobic preincubation of the wild-type strain, and in low-phosphate cells). The presence of NH₄⁺ and some amino acids (e.g., leucine, aspartate, cysteine and methionine) depressed the stimulation while that of cationic amino acids (typically arginine and lysine) and of K⁺ increased it by 50–80%. The stimulated (i.e., newly synthesized) transport system was degraded with a half-life of about 10 min.     

Infinite cis influx of cyclic AMP into human erythrocyte ghosts

Infinite cis uptake of cyclic AMP into red blood cell ghosts has been measured. The $\text{K}{}_{\mathrm{<mtext>i</mtext><mtext>c</mtext>}}{}^{\mathrm{<mtext>oi</mtext>}}$ is calculated from two different integrated rate equations that are applicable when the substrate concentration is unsufficient to cause volume changes. Values of 0.69 mM and 0.66 mM are obtained for the infinite cis$\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ at 30°C using these procedures. These values are only slightly higher than that predicted from zero trans net flux experiments.Lowering the temperature reduces $\text{K}{}_{\mathrm{<mtext>i</mtext><mtext>c</mtext>}}{}^{\mathrm{<mtext>oi</mtext>}}$ from 0.69 mM at 30°C to 0.478 mM at 20°C, 0.108 mM at 10°C and 0.072 mM at 4°C ($\text{Q}{}_{10}\text{= 2.4}$). The $\text{Q}{}_{10}$ for activation of influx permeability of 10^?5 M cyclic AMP is 1.55.     

Proton electrochemical gradient and phosphate potential in submitochondrial particles

1. The aerobic uptake of inorganic ions, such as ⁸⁶Rb⁺ or ¹²⁵I^?, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of ΔpH, the transmembrane pH differential, and Δψ, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the ΔpH and Δψ values from organic and inorganic ions is large at high but not at low ion/protein ratios.2. In the absence of weak bases and strong acids the values of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$, the proton electrochemical potential difference, are close to 100 mV and the magnitude of ΔpH and Δψ are similar. Weak bases decrease ΔpH and enhance Δψ. Strong acids decrease Δψ and enhance ΔpH. Interchangeability of ΔpH with Δψ occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$.3. Concentrations of weak bases capable of abolishing ΔpH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing Δψ affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of ΔpH + Δψ inhibit ATP synthesis.4. Depression of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ is paralleled by inhibition of ATP synthesis and decline of ΔGp, the phosphate potential. Abolition of ATP synthesis occurs only when $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ is below 20 mV. The $\text{ΔG}\text{p}\text{/Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ ratio increases hyperbolically with the decrease of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$.     

The role of the sites for ATP of the Ca2+-ATPase from human red cell membranes during Ca2+-phosphatase activity

1. In the presence of ATP, the Ca²⁺ pump of human red cell membranes catalyzes the hydrolysis of $\text{p-}\text{nitrophenyl}$ phosphate. The requirement for ATP of the Ca²⁺-$\text{p-}\text{nitrophenylphosphatase}$ activity was studied in relation to the two classes of site for ATP that are apparent during Ca²⁺ -ATPase activity. 2. (a) The $\text{K}{}_{0.5}$ for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ extrapolated at 0 mM PNPP is equal to the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the Ca²⁺ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca²⁺ -ATPase or as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 4. At ATP concentrations that almost saturate the high-affinity site, Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity increases as a function of PNPP along an S-shaped curve, while Ca²⁺ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca²⁺ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca²⁺ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca²⁺ -ATPase activity at the high-affinity size persists during Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity.     

Soluble and membrane-bound thiamine-binding proteins from Saccharomyces cerevisiae

Previous communications from this laboratory have indicated that there exists a thiamine-binding protein in the soluble fraction of Saccharomyces cerevisiae which may be implicated to participate in the transport system of thiamine in vivo.In the present paper it is demonstrated that both activities of the soluble thiamine-binding protein and thiamine transport in S. cerevisiae are greatest in the early-log phase of the growth and decline sharply with cell growth. The soluble thiamine-binding protein isolated from yeast cells by conventional methods containing osmotic shock treatment appeared to be a glycoprotein with a molecular weight of 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of the binding for thiamine was 29 nM which is about six fold lower than the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (0.18 μM) of thiamine transport. The optimal pH for the binding was 5.5, and the binding was inhibited reversibly by 8 M urea but irreversibly by 8 M urea containing 1% 2-mercaptoethanol. Several thiamine derivatives and the analogs such as pyrithiamine and oxythiamine inhibited to similar extent both the binding of thiamine and transport in S. cerevisiae, whereas thiamine phosphates, 2-methyl-4-amino-5-hydroxymethylpyrimidine and $\text{O-}\text{benzoylthiamine}$ disulfide did not show similarities in the effect on the binding and transport in vivo. Furthermore, it was demonstrated by gel filtration of sonic extract from the cells that a thiamine transport mutant of S. cerevisiae (PT-R₂) contains the soluble binding protein in a comparable amounts to that in the parent strain, suggesting that another protein component is required for the actual translocation of thiamine in the yeast cell membrane. On the other hand, the membrane fraction prepared from S. cerevisiae showed a thiamine-binding activity with apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 0.17μM at optimal pH 5.0 which is almost the same with the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the thiamine transport system. The membrane-bound thiamine-binding activity was not only repressible by exogenous thiamine in the growth medium, but as well as thiamine transport it was markedly inhibited by both pyrithiamine and $\text{O-}\text{benzoylthiamine}$ disulfide. In addition, it was found that membrane fraction prepared frtom PT-R₂ has the thiamine-binding activity of only 3% of that from the parent strain of S. cerevisiae.These results strongly suggest that membrane-bound thiamine-binding protein may be directly involved in the transport of thiamine in S. cerevisiae.     

An overall radioassay for the first three reactions of de novo pyrimidine biosynthesis

A sensitive radioassay is described for the overall biosynthetic activity of the multienzymatic protein which catalyzes the first three reactions of de novo pyrimidine biosynthesis in mammals. The ability of the multienzymatic protein to synthesize dihydroorotate can be assayed using $\text{[}{}^{14}\text{C]HCO}{}_3{}^{\mathrm{?}}$, l-[¹⁴C]aspartate, or [${}^{14}\text{C}$] carbamyl phosphate as substrate. The synthesis of the final product, l-dihydroorotate, may be coupled to synthesis of orotidine 5′-monophosphate to overcome the unfavorable equilibrium existing between l-dihydroorotate and its precursor, N-carbamyl-l-aspartate, in the physiological pH range (Christopherson, R. I., and Jones, M. E., 1979, J. Biol. Chem.254, 12506–12512). l-Aspartate and all pyrimidine intermediates from carbamyl phosphate to orotidine 5′-monophosphate can be clearly separated by ion-exchange chromatography in a single dimension on polyethyleneimine-cellulose chromatograms and carbamyl phosphate and its degradation product cyanate may be quantitated directly along with the other intermediates.     

Glucose transport in nongrowing yeast

The inducible, nonenergy-requiring glucose transport system of the yeast Kluyveromyces lactis is inactivated upon starving cells of glucose by (1) transferring logarithmic phase glucose-grown cells to synthetic medium containing a nonglycolytic carbon source, and (2) upon transition of logarithmic phase glucose-grown cells to stationary phase. The steady-state accumulation of nonmetabolizeable 6-deoxyglucose and the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of transport of 6-deoxyglucose is the same in stationary phase cells and in logarithmic phase cells. The rate of transport is lower in the nongrowing cells. Restoration of activity requires energy and protein synthesis as well as inducer.     

Inhibition of anion transport in human red blood cells by 5,5′-dithiobis(2-nitrobenzoic acid)

The uptake of [³²P]phosphate into human red blood cells was inhibited ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.6}\text{mM}$) 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 ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7}\text{mM}$). 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 ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 1?2 μ}\text{M}$), 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.     

Animal variation in alanine uptake by rabbit ileal mucosa

Alanine uptake by rabbit ileal mucosa has been measured in the presence and absence of Na⁺ to establish the characteristics of biological variation. Common $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values, calculated for two systems of alanine entry, were used for estimation of two J_max values for individual rabbits. The distribution of and within the population was normal and there was minimal interaction between these two parameters, judged by a cross-correlation analysis. Ways of processing data to account for this type of animal variation are discussed.     

Characteristics of leucine transport by isolated hepatocytes of antarctic fish at low temperatures

Hepatocytes prepared by collagenase perfusion from Antarctic nototheniid fish of genus Trematomus are active in uptake of [¹⁴C]leucine at 0, 5, and 10°C. The system is saturable with apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ about 1.0 mM. Isoleucine and phenylalanine were major competitors, valine was about one-half as effective, while alanine, glycine and histidine had no effect. Temperature dependency of rates in the 0–10°C range yielded $\text{E}{}_{\mathrm{<mtext>a</mtext>}}\text{= 65}\text{kJ/mol}$ ($\text{Q}{}_{10}\text{= 2.7}$). The average first-order rate constant at 0°C was 0.1 min^?1, one-third the value of 0.3 min^?1 estimated for clearance of [¹⁴C]leucine by liver of these species in vivo. Affinity and specificity agreed well with in vivo data on liver clearance of leucine, both in Antarctic fish at 0°C and in temperate fish acclimated to 10°C and 20°C. The results indicate similar modifications of leucine transport associated with evolutionary cold adaptation and seasonal acclimation in fish.     

Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish

Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca²⁺-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na⁺ gradient ($\text{out > in}$). In addition, amino acid uptake requires Cl^? in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na⁺- and Cl^?-dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl^? requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl^?. In the presence of Cl^?, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 2-aminoisobutyric acid uptake is reduced and its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ is enhanced. Cl^? affects also a non-saturable Na⁺-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl^? (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient ($\text{in > out}$) as well as the presence of Na⁺ in the incubation medium seems to enter into this requirement. This intravesicular Cl^? effect is not influenced by pH between 6.5 and 8.4.     

Reconstitution into liposomes of glucose active transport from the rabbit renal proximal tubule. Characteristics of the system

This paper describes the characteristics of Na⁺-dependent d-glucose transport into liposomes made from soybean phospholipids into which have been reconstituted detergent-solubilized components from the rabbit renal proximal tubular brush border membrane. Conditions for optimal and quantitative reconstitution of glucose carriers are defined. Na⁺-dependent d-glucose uptake occurs via a saturable system with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.125–0.135 mM, is responsive to the volume of the internal liposomal space, and shows ‘overshoot’ as seen in natural membranes. The rate of Na⁺-dependent d-glucose uptake and the magnitude of the ‘overshoot’ are proportional to the concentration of protein used in reconstitution.