Sugar Uptake and Metabolism in the Developing Endosperm of Tassel-seed Tunicate (Ts-5 Tu) Maize

Factors regulating assimilate transport into developing maize (Zea mays L.) kernels have been difficult to determine because of the structural complexity of basal kernel tissues and the damage that results from tissue dissection. The sensitivity of maize kernels to experimental manipulation is such that substantial maternal tissue is required to support kernel growth in vitro. Consequently, sugar transport experiments with isolated seed tissues or detached kernels have not unequivocally demonstrated how sugar transport occurs. In the present study, Tassel-seed Tunicate (Ts-5 Tu) maize kernels were investigated as a model system for introducing test solutions into the pedicel apoplast with minimal wounding. Transpiration in leafy glumes drew ¹⁴C-sugar solutions up the 8- to 10-millimeter-long pedicel stalks into the basal endosperm transfer cell region. ¹⁴C from fructose was incorporated into starch for 8 days. Sugar uptake into endosperm and embryo tissue showed specificity and inhibitor sensitivity. In particular, p-chloromercuribenzene sulfonate partially inhibited fructose uptake into the endosperm but had no effect on the metabolic conversion of that fructose that entered the endosperm. These results are consistent with active, carrier-mediated sugar transport, but a definitive determination would require more detailed tissue analysis. We propose that further refinement of the incubation solution may allow long-term kernel growth without cob tissue and thus provide a more precise determination of which maternal factors influence seed development.     

Transport of sugars into microvessels isolated from rat brain: a model for the blood-brain barrier.

Abstract— Microvessels (primarily capillaries) were isolated from the brains of rats 25-35 days of age. This preparation was characterized by light, transmission, and scanning electron microscopy. Transmission electron microscopy revealed that the endothelial cell membranes were intact and were impermeable to horseradish peroxidase. However, scanning electron microscopy revealed that damage to the membrane occurred during isolation. The isolated microvessel preparations were metabolically competent as demonstrated by their ability to metabolize [¹⁴C]glucose. Aliquots of microvessel preparation were incubated with radioactive non-metabolizable analogs of D-glucose at various concentrations. The kinetics of accumulation of radioactivity in the capillaries were analyzed according to a model for carrier-mediated diffusion and affinity constants for 3-O-methyl- D-glucose and 2-deoxyglucose were calculated (about 18 mM at 20°C in each case). These affinity constants are somewhat greater than that expected from whole animal experiments reported by other laboratories. This discrepancy is probably accounted for by the presence of a passive diffusion component. However, despite this complication, the primary mechanism for entry of D-glucose analogues at physiological concentrations is compatible with carrier-mediated transport since: the uptake of sugar analogs was shown to be saturable, to exhibit competition for uptake between structurally similar molecules, and to be non-concentrative. In contrast, the uptake of glycerol, mannitol, and L-glucose by isolated microvessels obeyed the kinetics of simple passive diffusion and was not saturable. Our results are compatible with the concept that the capillary is the anatomic locus of the blood-brain barrier and that this structure contains the carrier-mediated transport system for monosaccharide penetration into brain.     

Membrane Potential Changes Related to Active Transport of Glycine in Lemna gibba G1

Accumulation of ¹⁴C-labeled glycine and microelectrode techniques were employed to study glycine transport and the effect of glycine on the membrane potential (Δψ) in Lemna gibba G1. Evidence is presented that two processes, a passive uptake by diffusion and a carrier-mediated uptake, are involved in glycine transport into Lemna cells. At the onset of active glycine uptake the component of Δψ which depended on metabolism was decreased. The depolarized membrane repolarized in the presence of glycine. This glycine-induced depolarization followed a saturation curve with increasing glycine concentration which corresponded to carrier-mediated glycine influx kinetics. The transport of glycine was correlated with the metabolically dependent component of Δψ. It is suggested (a) that the transient change in Δψ reflects the operation of an H⁺-glycine cotransport system driven by an electrochemical H⁺ gradient; and (b) that this system is energized by an active H⁺ extrusion. Therefore the maximum depolarization of the membrane consequently depended on both the rate of glycine uptake and the activity of the proton extrusion pump.     

Glucose Transport in the Malarial (Plasmodium lophurae) Infected Erythrocyte*

Plasmodium lophurae-infected red blood cells utilized considerably greater quantities of glucose than did uninfected duckling red cells. Kinetic analysis of glucose transport showed: (A). Below a concentration of 2 mM in the medium the uptake process followed Michaelis-Menten kinetics (carrier-mediated facilitated diffusion) whereas at concentrations greater than this simple diffusion became the main mode of entry. (B). The apparent transport constants, K_t, for normal and infected cells were similar. However there was an 8-fold increase in the maximal velocity, V_max, for infected cells. (C). “Free” malaria parasites had a significantly lower K_t and a higher V_max than did normal or infected red cells. Entry and exit studies with the nonmetabolizable sugar analog, 3-0-methyl glucose, demonstrated that the enhanced rate of uptake by infected cells involved an increase in the simple diffusion component and the degree of enhancement was correlated with the size of the intracellular parasite. Competition experiments suggested that in the malaria-infected cell one locus is involved in the carrier-mediated transport of glucose, mannose and galactose whereas another locus transports fructose and/or glycerol. These results indicate that the enhanced entry of glucose into the malaria-infected red cell is a consequence of factors other than increased glucose catabolism by the host-parasite complex, and the host cell's capacity to take up greater quantities of sugar directly involves the growing intracellular plasmodium.     

Membrane function in differentiating skeletal muscle cells: I. Kinetic analysis of amino acid transport

Primary cultures of mononucleated myoblasts from 12-day-old chick embryos have a twofold higher rate of α-aminoisobutyric acid (AIB) transport before fusion occurs to form multinucleated myotubes. Several lines of evidence indicate that the uptake of AIB observed in both myoblasts and myotubes is primarily carrier-mediated by a membrane transport system. Increasing the temperature from 24 to 37°C results in a threefold increase in the rate of AIB uptake; both methionine and glycine inhibit AIB uptake by more than 85%; and 2,4-dinitrophenol inhibits AIB uptake by approximately 50%. In addition, the energies of activation (14.5 and 14.0 kcal/mole for myoblasts and myotubes, respectively) are characteristic of carrier-mediated transport. Resolution of AIB uptake into a saturable, carrier-mediated component and a nonsaturable, diffusion component shows that at concentrations of AIB≤1.5 mM over 97% of total AIB uptake is carriermediated in both myoblasts and myotubes. Kinetic analysis of carrier-mediated AIB uptake indicates that myoblasts and myotube membrane carriers have the same affinity for AIB (K_m values = 1.73 and 1.31 mM, respectively). However, the V_max for myoblasts is 23.7 nmole/mg/min while myotubes have a V_max of 12.6 nmole/mg/min. The twofold difference in V_max is shown to be due to a twofold difference in the quantity of membrane transport sites per milligram of protein.     

Role of Ca2+ and Mg2+ in neutrophil hexose transport

The influence of extracellular Ca²⁺ and Mg²⁺ on the transport of 2-deoxy-[³H]glucose into human polymorphonuclear neutrophils was studied. Omission of these cations from the cell suspensions had little effect on resting hexose uptake. Furthermore, the addition of the bivalent cation chelator, EDTA, depressed uptake only slightly. Similarly, neither cation was essential for the enhanced 2-deoxy-D-[³H]glucose uptake stimulated by two chemotactic factors (C5a and $\text{N-}\text{formylmethionylleucylphenylalanine}$) and arachidonic acid: enhanced uptake was only partially depressed by the omission of Ca²⁺ and Mg²⁺ from the suspensions and was still prominent in the presence of EDTA. Two other neutrophil stimulants, the ionophores, A23187 and ionomycin, also enhanced hexose uptake but their actions were heavily dependent upon extracellular bivalent cations and were totally abrogated by EDTA. In all instances, extracellular Ca²⁺, but not Mg²⁺, supported optimal enhanced hexose transport induced by stimuli.Activation of 2-deoxy-D-[³H]glucose uptake by each of the five stimuli was totally blocked by cytochalasin B (a blocker of carrier-mediated hexose transport) and D-glucose but not by L-glucose. The data indicate, therefore, that a variety of neutrophil stimulants activate carrier-mediated hexose transport. Although this transport can be triggered by the movement of extracellular Ca²⁺ into the cell (as exemplified by the action of the two ionophores), such Ca²⁺ movement is not required for the actions of chemotactic factors or arachidonic acid. Other mechanisms, such as a rearrangement of intracellular Ca²⁺, may be involved in mediating the activation of hexose transport induced by the latter stimuli.     

Choline influx across the brush border of guinea pig jejunum

Choline uptake across the mucosal border of guinea pig jejunum was measured to determine the characteristics of this step in intestinal absorption. Unidirectional influx of [¹⁴C]choline appears to proceed primarily by a saturable, carrier-mediated process at low mucosal choline concentrations; at high concentrations (>4 mM) the influx rate is approximately linearly related to the mucosal choline concentration, suggesting that absorption by passive diffusion predominates. Influx was only minimally reduced by elimination of Na⁺ from the mucosal test solution or by reduction of the intracellular Na⁺ concentration. Preincubation of tissue samples with metabolic inhibitors or with ouabain did not markedly reduce influx. These results are consistent with a model of choline transport across the brush border membrane by a carrier-mediated mechanism which is similar to that involved in fructose absorption but different from the Na⁺-dependent mechanism which participates in active transport of sugar and amino acids. At low lumenal choline concentrations, influx into colonic mucosa is slower than in jejunum and appears to be attributed solely to simple diffusion.     

The role of glucose and pyruvate transport in regulating nutrient utilization by preimplantation mouse embryos

Preimplantation mouse embryos utilize pyruvate preferentially during the early cleavage stages before switching to glucose at around the time of compaction. This switch in substrate preference has been studied using a non-invasive ultramicrofluorometric analytical technique on single mouse embryos. On the basis of transport kinetic studies and inhibition by phloretin, cytochalasin B and sugar analogues, a component of glucose uptake by mouse blastocysts was found to be mediated by facilitated diffusion. The Jmax and Kt of this facilitated component were 3.53 pmol embryo-1 h-1 and 0.14 mM, respectively. At physiological concentrations of glucose, the facilitated component accounts for around 75% of glucose uptake. Glucose uptake by blastocysts was found to be insensitive to insulin, added at a range of concentrations. There was no evidence for glucose active transport. The carrier-mediated component of glucose entry was detectable from the 2-cell stage onwards. Pyruvate uptake was also mediated by a carrier throughout development. In the absence of glucose in the incubation medium, the characteristic decline in pyruvate uptake does not occur. The data are consistent with a role for embryonic cell transport in regulating glucose utilization prior to compaction, but do not exclude the involvement of metabolic factors, such as the allosteric regulation of the enzymes hexokinase and phosphofructokinase.     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H⁺-dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of d- and l-[U-¹⁴C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-d-glucose/d-[U-¹⁴C]glucose and 3-O-methyl-d-glucose/3-O-methyl-d-[U-¹⁴C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH₄Cl inhibited neither the linear component of d- and l-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-d-[U-¹⁴C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol^− 1, respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol^− 1). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

Nitrate Utilization by the Diatom Skeletonema costatum: II. Regulation of Nitrate Uptake

Nitrate utilization has been characterized in nitrogen-deficient cells of the marine diatom Skeletonema costatum. In order to separate nitrate uptake from nitrate reduction, nitrate reductase activity was suppressed with tungstate. Neither nitrite nor the presence of amino acids in the external medium or darkness affects nitrate uptake kinetics. Ammonium strongly inhibits carrier-mediated nitrate uptake, without affecting diffusion transfer. A model is proposed for the uptake and assimilation of nitrate in S. costatum and their regulation by ammonium ions.     

Galactose Transport in Saccharomyces cerevisiae III. Characteristics of Galactose Uptake in Transferaseless Cells: Evidence Against Transport-Associated Phosphorylation

The characteristics of the inducible galactose transport system in bakers' yeast were studied in uridine diphosphate, galactose-1-phosphate uridylyl-transferaseless cells. Transferaseless cells transport galactose at the same initial rate as wild-type cells and accumulate a mixture of free galactose and galactose-1-phosphate. The addition of ¹⁴C-labeled galactose to cells preloaded with unlabeled galactose and galactose-1-phosphate results in a higher rate of labeling of the free-sugar pool than of the galactose-1-phosphate pool. These results support other evidence that galactose uptake in bakers' yeast is a carrier-mediated, facilitated diffusion and that phosphorylation is an intracellular event after uptake of the free sugar.     

The mechanism of sugar uptake by sugarcane suspension cells

Sugarcane cell suspensions took up sugar from the medium at rates comparable to or greater than sugarcane tissue slices or plants in the field. This system offers an opportunity for the study of kinetic and energetic mechanisms of sugar transport in storage parenchyma-like cells in the absence of heterogeneity introduced by tissues. The following results were obtained: (a) The sugar uptake system was specific for hexoses; as previously proposed, sucrose was hydrolyzed by an extracellular invertase before the sugar moieties were taken up; no evidence for multiple sugar uptake systems was obtained. — (b) Uptake of the glucose-analog 3-O-methylglucose (3-OMG) reached a plateau value with an intracellular concentration higher than in the medium (approximately 15-fold). — (c) There was a balance of influx and efflux during steady state; the rate of exchange influx was lower than the rate of net influx; the K_m value was higher (70 M) than for net influx (24 M); the exchange efflux is proposed to be mediated by the same transport system with a K_m value of approximately 2.6 mM for internal 3-OMG; the rate of net efflux of hexoses was less than a third of the rate of exchange efflux. — (d) The uptake of hexoses proceeded as proton-symport with a stoichiometry of 0.87 H⁺ per sugar; during the onset of hexose transport there was a K⁺ exit of 0.94 K⁺ per sugar for charge compensation. (It was assumed that the real stoichiometries are 1 H⁺ and 1 K⁺ per sugar.) The K_m values for sugar transport and sugar-induced proton uptake were identical. Sucrose induced proton uptake only in the presence of cell wall invertase. — (e) There was no net proton uptake with 3-OMG by cells which were preloaded with glucose though there was significant sugar uptake. It is assumed, therefore, that the exit of hexose occurs together with protons. — (f) The protonmotive potential of sugarcane cells corresponded to about 120 mV: pH-gradient 1.1 units, membrane potential of-60 mV (these values increased if vacuolar pH and membrane potential were also considered). It was abolished by uncouplers, and the magnitude of the components depended on the external pH value. We present evidence for the operation of a proton-coupled sugar transport system in cell suspensions that were derived from, and have characteristics of, storage parenchyma. The quantitative rates of sugar transport suggest that the role of this transport system is not limiting for sugar storage.Abbreviations 3-OMG 3-O methylglucose - DMO 5,5-dimethyl-2, 4-oxazolidinedione - TPP tetraphenylphosphonium chloride - CCCP carbonyl cyanide, m-chlorophenylhydrozane     

Sucrose uptake by developing soybean cotyledons

Sucrose uptake by excised developing soybean cotyledons shows a biphasic dependence on sucrose concentration. At concentrations less than about 50 millimolar external sucrose, uptake can be described as a carrier-mediated process, with a K_m of 8 millimolar. At higher external sucrose concentrations, a linear dependence becomes apparent, which suggests the participation of a nonsaturable component in total uptake. Sucrose absorption is dependent on the presence of an electrochemical potential gradient for protons since agents interfering with the generation or maintenance of this gradient (NaN₃ or carbonylcyanide-m-chlorophenyl hydrazone) decrease sucrose transport to a level at or below that predicted from the operation of the noncarrier-mediated process alone. The saturable component of sucrose uptake is also sensitive to the sulfhydryl-modifying compounds N-ethylmaleimide and p-chloro-mercuribenzenesulfonate. The thiol-reducing agent diethioerythritol reverses fully the p-chloro-mercuri-benzenesulfonate inhibition, but not that of N-ethyl maleim de. Sucrose transport is sensitive to external pH, being decreased at high pH₀. Since sucrose-induced depolarization of the membrane potential and carrier-mediated sucrose influx show similar pH-dependence, inhibitor sensitivity, and values of K_m for sucrose, a sucrose/proton contransport process appears to operate in developing soybean cotyledon cells. Measurement of free space and intracellular sucrose concentrations in vivo suggests that the carrier-mediated process is fully saturated and that sucrose transport may be limiting for sucrose accumulation by the developing seed.     

3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

The characteristics and regulatory nature of sugar transport in freshly isolated bovine adrenal chromaffin cells were investigated. Transport was measured by following the cell/medium distribution of non-metabolizable glucose analogue, 3-O-methyl-D-glucose. The uptake of 3-O-methyl-D-glucose was was mediated by a saturable transport system with a Km of 8.2 mM and a Vmax of 0.69 nmol/mg protein per min. Basal 3-O-methyl-D-glucose transport was competitively inhibited by D-glucose and a countertransport effect was demonstrated. Cytochalasin B and phloretin, which are specific inhibitors of carrier-mediated glucose transport, significantly decreased basal 3-O-methyl-D-glucose uptake. Basal transport was stimulated by 50 mU/ml insulin, an effect associated with an increase in Vmax. The stimulatory effect of insulin was depressed in medium lacking external Ca2+, or containing the Ca2+-antagonistic ion, La3+, or the Ca2+ channel blocker, methoxyverapamil (D-600). The data suggest that the uptake of 3-O-methyl-D-glucose in freshly isolated bovine adrenal chromaffin cells is mediated by a specific facilitated diffusion mechanism, and is subject to regulation by insulin, thus resembling sugar transport in muscle. In addition, the insulin effect appears to depend on the presence of extracellular Ca2+.     

Potassium transport in Neurospora. Evidence for a multisite carrier at high pH

At low extracellular pH (4–6), net uptake of potassium by Neurospora is a simple exponential process which obeys Michaelis kinetics as a function of [K]_o. At high pH, however, potassium uptake becomes considerably more complex, and can be resolved into two distinct exponential components. The fast component (time constant = 1.2 min) is matched quantitatively by a rapid loss of sodium; it is attributed to ion exchange within the cell wall, since it is comparatively insensitive to low temperature and metabolic inhibitors. By contrast, the slower component (time constant = 10.9 min) is inhibited markedly at 0°C and by CN and deoxycorticosterone, and is thought to represent carrier-mediated transport of potassium across the cell membrane. This transport process exhibits sigmoid kinetics as a function of [K]_o; the data can be fitted satisfactorily by two different two-site models (one involving a carrier site and a modifier site, the other an allosteric model). Either of these models could also accommodate the simple Michaelis kinetics at low pH.     

In Vitro Sugar Transport in Zea mays L. Kernels : I. Characteristics of Sugar Absorption and Metabolism by Developing Maize Endosperm

Short-term transport studies were conducted using excised whole Zea mays kernels incubated in buffered solutions containing radiolabeled sugars. Following incubation, endosperms were removed and rates of net ¹⁴C-sugar uptake were determined. Endogenous sugar gradients of the kernel were estimated by measuring sugar concentrations in cell sap collected from the pedicel and endosperm. A sugar concentration gradient from the pedicel to the endosperm was found. Uptake rates of ¹⁴C-labeled glucose, fructose, and sucrose were linear over the concentration range of 2 to 200 millimolar. At sugar concentrations greater than 50 millimolar, hexose uptake exceeded sucrose uptake. Metabolic inhibitor studies using carbonylcyanide-m-chlorophenylhydrazone, sodium cyanide, and dinitrophenol and estimates of Q₁₀ suggest that the transport of sugars into the developing maize endosperm is a passive process. Sucrose was hydrolyzed to glucose and fructose during uptake and in the endosperm was either reconverted to sucrose or incorporated into insoluble matter. These data suggest that the conversion of sucrose to glucose and fructose may play a role in sugar absorption by endosperm. Our data do not indicate that sugars are absorbed actively. Sugar uptake by the endosperm may be regulated by the capacity for sugar utilization (i.e. starch synthesis).     

Kinetics of manganese transport and gene expressions of manganese transport carriers in Caco-2 cell monolayers

Two experiments were conducted to investigate the kinetics of manganese (Mn) transport in Caco-2 cell monolayers and the gene expressions of Mn transport carriers in apical (AP) and basolateral (BL) membranes. In experiment 1, the cells were treated with the medium containing 146 μmol/L of Mn (MnSO₄·H₂O). Both the uptake and transport of Mn from AP–BL or from BL–AP at different time-points were assessed to determine the optimal time for kinetics of Mn transport. The transport of Mn increased linearly with higher efficiency values in AP–BL than in BL–AP direction, however, the uptake of Mn revealed an asymptotic pattern within 120 min. In experiment 2, the kinetics of Mn transport in AP–BL was determined with media containing Mn concentrations from 0 to 2,500 μmol/L at 40 and 120 min, respectively, and mRNA levels of divalent metal transporter 1 (DMT1) and ferroportin (FPN1) were determined in Caco-2 cells treated with the medium containing 0 or 800 μmol/L of Mn for 120 min. The kinetics of Mn transport showed a carrier-mediated process when Mn concentrations were lower than 1,000 μmol/L and a linear increment when Mn concentrations exceeded 1,000 μmol/L at either 40 or 120 min. Mn treatment decreased (P < 0.01) DMT1 mRNA level and increased (P < 0.01) FPN1 mRNA level. The results from the present study suggested that Mn transport in AP–BL fit both carrier-mediated saturable and non-saturable diffusion processes, and Mn transport carriers DMT1 and FPN1 mediate the apical uptake and basolateral exit of Mn in Caco-2 cells.     

The rate of uptake of carbon monoxide and of nitric oxide by normal human erythrocytes and experimentally produced spherocytes

The uptake of gases such as oxygen, carbon monoxide, or nitric oxide by the erythrocyte involves: (a) diffusion across the cellular membrane, (b) intraerythrocytic diffusion, and (c) chemical combination with hemoglobin. The aim of this investigation was to obtain data which would permit an analysis of each of these factors in limiting the rate of gas uptake. The initial over-all rate of uptake of gases which combine chemically with hemoglobin to produce a color change can be measured by a modified version of the Hartridge-Roughton-Millikan constant flow, rapid reaction apparatus. If nitric oxide is the reactant gas, only (a) and (b) are measured since the chemical combination of this gas with hemoglobin is extremely rapid. Our studies have shown that human biconcave discoidal erythrocytes at 38 and 48°C., have the same initial rate of carbon monoxide and nitric oxide uptake as the same cells converted into spherocytes of equal volume. Similarly there was no difference between discs and cells sphered with a 30 per cent increase in volume. Shrunken erythrocytes showed a marked decrease in rate of gas uptake. This suggests that surface area and maximum linear distance for intracellular diffusion of this magnitude do not measurably retard gas uptake. In the shrunken cells, a change in the orientation and concentration of intraerythrocytic hemoglobin and/or of the membrane components may have impeded gas diffusion.     

Mechanisms for the facilitated diffusion of substrates across cell membranes

Two classes of theoretical mechanisms for protein-mediated, passive, transmembrane substrate transport (facilitated diffusion) are compared. The simple carrier describes a carrier protein that exposes substrate influx and efflux sites alternately but never both sites simultaneously. Two-site models for substrate transport describe carrier proteins containing influx and efflux sites simultaneously. Velocity equations describing transport by these mechanisms are derived. These equations take the same general form, being characterized by five experimental constants. Simple carrier-mediated transport is restricted to hyperbolic kinetics under all conditions. Two-site carrier-mediated transport may deviate from hyperbolic kinetics only under equilibrium exchange conditions. When both simple- and two-site carriers display hyperbolic kinetics under equilibrium exchange conditions, these models are indistinguishable by using steady-state transport data alone. Seven sugar transport systems are analyzed. Five of these systems are consistent with both models for sugar transport. Uridine, leucine, and cAMP transport by human red cells are consistent with both simple- and two-site models for transport. Human erythrocyte sugar transport can be modeled by simple- and two-site carrier mechanisms, allowing for compartmentalization of intracellular sugars. In this instance, resolution of the intrinsic properties of the human red cell sugar carrier at 20 degrees C requires the use of submillisecond transport measurements.     

Solubilization and reconstitution of the glucose transport system from Saccharomyces cerevisiae

The glucose transport system from Saccharomyces cerevisiae was solubilized from isolated plasma membranes by the nonionic detergent, octylglucoside. The transport system was reconstituted into proteoliposomes with removal of detergent from the extract by dialysis, followed by the addition of asolectin liposomes to the dialyzed proteins with a freeze-thaw and brief bath-sonication step. The reconstituted proteoliposomes exhibit specific carrier-mediated facilitated diffusion of d-glucose, including stimulated equilibrium exchange and influx counterflow. Furthermore, the reconstituted facilitated diffusion system shows substrate specificities similar to those of the intact cell d-glucose transport system.