Dependent D-glucose Uptake Research Articles

Glucose transport during Ageing by human intestinal brush-border membrane vesicles

Glucose absorption by the small intestine is a complex phenomenon, that can be successfully studied by means of isolated brush-border and basolateral membrane vesicles of the enterocytes. We have carried out transport experiments on isolated brush-border membrane vesicles from the human small intestine, taking into account the age of the subjects. Our studies demonstrated that Na +-glucose co-transporter activity decreased as age increased with the “overshoot” phenomenon disappearing altogether in the oldest subjects. This effect was compared to that observed in intestinal membrane vesicles of young patients suffering from Crohn's disease; in this case there was a marked decrease in the Na +-dependent d-glucose uptake, but the “overshoot”, even though low, was present. K +-dependent d-glucose transport, diffusion of l-glucose and the levels of some enzyme markers for intestinal brush-border membranes were also studied.

Monoclonal antibodies against the renal Na+-D-glucose cotransporter. Identification of antigenic polypeptides and demonstration of functional coupling of different Na+-cotransport systems.

Eight monoclonal antibodies are described which are directed against the renal Na+-D-glucose cotransporter. In porcine renal brush-border membranes, the antibodies either bind to one or to three polypeptides which have been identified as components of the Na+-D-glucose cotransporter (Neeb, M., Kunz, U., and Koepsell, H., (1987) J. Biol. Chem. 262, 10718-10727). Their molecular weights and isoelectric points are 75,000 and pH 5.5, 60,000 and pH 5.2, and 47,000 and pH 5.4. Six antibodies were able to influence Na+-dependent D-glucose uptake and/or Na+-dependent high affinity phlorizin binding. In the presence of Na+, the binding of all antibodies to native membrane proteins was altered by D-glucose but not by D-mannose. Since this effect was observed with D-glucose concentrations less than 1 x 10(-8) M, a high affinity D-glucose-binding site on the D-glucose transporter has been implied. Some of the antibodies probably interact also with other Na+-coupled transporters since their binding was altered by micromolar concentrations of L-lactate, L-alanine, or L-glutamate but not by the nontransported control substances D-alanine and D-glutamate. L-lactate increased the binding of one antibody in the absence but not in the presence of D-glucose. Effects of L-lactate and L-alanine on the binding of another antibody were only observed when D-glucose was present. Thus, some epitopes on the Na+-D-glucose cotransporter are altered by D-glucose and also by substrates of other Na+ cotransporters. This finding suggests functional coupling of different Na+-cotransport systems.

Brush border membrane vesicles formed from human duodenal biopsies exhibit NA +-dependent concentrative L-leucine and D-glucose uptake

The human duodenum actively transports L-leucine and D-glucose under Na+ gradient conditions as demonstrated by uptake studies using brush border membrane vesicles from organ donor duodenum. Brush border membrane vesicles formed from peroral duodenal biopsies likewise demonstrate Na+ dependent concentrative uptake of D-glucose and L-leucine. This is the first demonstration of active transport processes in human duodenum. A simple microvesiculation method to form these vesicles is described as well as its potential application to clinical medicine in studying diseases of defective intestinal transport.

Radiation-inactivation studies on brush-border-membrane vesicles. General considerations, and application to the glucose and phosphate carriers.

Radiation-inactivation studies were performed on brush-border-membrane vesicles purified from rat kidney cortex. No alteration of the structural integrity of the vesicles was apparent in electron micrographs of irradiated and unirradiated vesicles. The size distributions of the vesicles were also similar for both populations. The molecular sizes of two-brush-border-membrane enzymes, alkaline phosphatase and 5'-nucleotidase, estimated by the radiation-inactivation technique, were 104800 +/- 3500 and 89,400 +/- 1800 Da respectively. Polyacrylamide-gel-electrophoresis patterns of membrane proteins remained unaltered by the radiation treatment, except in the region of higher-molecular-mass proteins, where destruction of the proteins was visible. The molecular size of two of these proteins was estimated from their mobilities in polyacrylamide gels and was similar to the target size, estimated from densitometric scanning of the gel. Intravesicular volume, estimated by the uptake of D-glucose at equilibrium, was unaffected by irradiation. Uptake of Na+, D-glucose and phosphate were measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Na+-independent D-glucose and phosphate uptakes were totally unaffected in the dose range used (0-9 Mrad). The Na+-dependent uptake of D-glucose was studied in irradiated vesicles, and the molecular size of the transporter was found to be 288,000 Da. The size of the Na+-dependent phosphate carrier was also estimated, and a value of 234,000 Da was obtained.

Preparation of basolateral membranes that transport p‐aminohippurate from primary cultures of rabbit kidney proximal tubule cells

The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.

Changes in glucose uptake by and phlorizin binding to brush-border membrane vesicles of small intestine from streptozotocin-induced diabetic rats.

The changes in the intestinal glucose transport in streptozotocin-induced diabetic rats have been demonstrated with brush-border membrane vesicles. When Na+-dependent D-glucose uptake and phlorizin binding activities were compared, both significantly increased either in the jejunum or ileum of diabetic rats compared with those of control. Kinetic studies showed the increases in the Vmax of glucose transport and the maximum binding of phlorizin (Bmax), whereas the Kt and Kd remained unchanged, respectively. These results suggested that the increase in glucose transport in diabetic rat intestine was due to the increase in the number of the intact glucose transporters.

Formula omitted] as a competitive inhibitor of Na +-dependent d-glucose cotransporter in the small intestine brush-border membrane

1-O-n- Octyl-β- d-glucopyranoside is a competitive inhibitor of the Na +-dependent d-glucose uptake into rabbit, rat and human intestinal brush-border membrane vesicles. The lack of effect on the equilibrium uptake demonstrates that the detergent does not act by rupturing the vesicles; no membrane leakiness was apparent at the concentrations of octylglucopyranoside used, since d-glucose uptake is not inhibited even in the absence of the Na + gradient (in K + solution). There is a competitive interaction between octylglucopyranoside and d-glucose, as shown by Dixon and by Hunter and Down plots. The selectivity of the detergent effect is confirmed by its modest influence on amino acid uptake.

Ischemia induces surface membrane dysfunction. Mechanism of altered Na+-dependent glucose transport.

Reversible ischemia reduced renal cortical brush border membrane (BBM) Na+-dependent D-glucose uptake (336 +/- 31 vs. 138 +/- 30 pmol/mg per 2 s, P less than 0.01) but had no effect on Na+-independent glucose or Na+-dependent L-alanine uptake. The effect on D-glucose uptake was present after only 15 min of ischemia and was due to a reduction in maximum velocity (1913 +/- 251 vs. 999 +/- 130 pmol/mg per 2 s; P less than 0.01). This reduction was not due to more rapid dissipation of the Na+ gradient, altered sidedness of the vesicles, or an alteration in membrane potential. Ischemia did, however, reduce the BBM sphingomyelin-to-phosphatidylcholine (SPH/PC) and cholesterol-to-phospholipid ratios and the number of specific high-affinity Na+-dependent phlorizin binding sites (390 +/- 43 vs. 146 +/- 24 pmol/mg; P less than 0.01) without altering the binding dissociation constant (Kd). 20 mM benzyl alcohol also reduced the number of Na+-dependent phlorizin binding sites (418 +/- 65 vs. 117 +/- 46; P less than 0.01) without altering Kd. The reduction in Na+-dependent D-glucose transport correlated with ischemic-induced changes in the BBM SPH/PC and cholesterol-to-phospholipid ratios and membrane fluidity. Taken together these data indicate the cellular site responsible for ischemic-induced reduction in renal cortical transcellular glucose transport is the BBM. We propose the mechanism involves marked alterations in BBM lipids leading to large increases in BBM fluidity which reduces the binding capacity of Na+-dependent glucose carriers. These data indicate that reversible ischemia has profound effects on the surface membrane function of epithelial cells.

Decreased transport of d-glucose and l-alanine across brush-border membrane vesicles from small intestine of rats treated with mitomycin C

To elucidate the mechanisms underlying the dysfunctions of intestinal absorption induced by antitumor drugs, the effect of pretreatment with mitomycin C on sodium gradient-dependent d-glucose and l-alanine transports was studied in rat brush-border membrane vesicles. 24, 48, 96, or 120 h following a single intravenous injection of mitomycin C, brush-border membrane vesicles were prepared from rat small-intestines. The uptake of d-glucose and l-alanine was shown to be Na + gradient-dependent even in the case of vesicles obtained from mitomycin C-treated rats, but uptake rates measured at 15 s and magnitude of overshooting effect in uptake of both solutes were decreased in vesicles maximally from 48 h mitomycin C-treated rats. The rate of d-glucose uptake calculated at 15 s recovered to the control level in vesicles prepared at 96 h and 120 h after mitomycin C-treatment, indicating that the effect of mitomycin C on Na + gradient-dependent d-glucose transport would be fully reversible. Tracer exchange experiments under Na + and d-glucose equilibrated conditions indicated that the Na +/ d-glucose transporters were similarly operative in the vesicles from control and 48 h mitomycin C-treated rats. Rates of 22Na + uptake measured at 15 s in vesicles from 48 h mitomycin C-treated rats, however, were increased. The increased permeability to Na + might bring about a more rapid dissipation of the Na + gradient in these vesicles and this would secondarily cause the decrease in Na +-dependent d-glucose uptake in vesicles from mitomycin C-treated rats.

Segmental localization of the rabbit renal proximal tubular Na+-H+ exchange system.

The activity of the Na+-H+ exchanger in rabbit proximal tubule was investigated by using luminal membrane vesicles prepared from "outer cortex" (proximal convoluted tubule) or "outer medulla" (proximal straight tubule). The purity of the preparations was examined by measuring the activity of several marker enzymes, and the degree of cross-contamination and the functional state of the membrane vesicles were assessed by studying Na+-dependent uptake of D-glucose. The Na+ uptake by pars convoluta membrane vesicles exhibited an overshoot in the presence of an intravesicular greater than extravesicular H+ gradient. The overshoot was eliminated by omitting or reversing the transmembranal H+ gradient or by adding amiloride. In contrast, Na+ uptake by pars recta membrane vesicles did not show an overshoot and was independent of H+ gradients and of amiloride. However, Na+ uptake by pars recta membrane vesicles pretreated with monensin exhibited an overshoot. This overshoot apparently was amiloride insensitive. The findings propose that the Na+-H+ exchanger is predominantly operative in the proximal convoluted tubule and is either lacking or of minor significance in the proximal straight tubule.

Renal transport of neutral amino acids. Tubular localization of Na+-dependent phenylalanine- and glucose-transport systems.

The transport properties for phenylalanine and glucose in luminal-membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit kidney were studied by a spectrophotometric method. Uptake of phenylalanine as well as of glucose by the two types of membrane vesicles was found to be Na+-dependent, electrogenic and stereospecific. Na+-dependent transport of L-phenylalanine by outer-cortical membrane vesicles could be accounted for by one transport system (KA congruent to 1.5 mM). By contrast, in the outer-medullary preparation, L-phenylalanine transport occurred via two transport systems, namely a high-affinity system with K1A congruent to 0.33 mM and a low-affinity system with K2A congruent to 7 mM respectively. Na+-dependent uptake of D-glucose by pars convoluta and pars recta membrane vesicles could be described by single, but different, transport systems, namely a low-affinity system with KA congruent to 3.5 mM and a high-affinity system with KA congruent to 0.30 mM respectively. Attempts to calculate the stoichiometry of the different Na+/D-glucose transport systems by using Hill-type plots revealed that the ratio of the Na+/hexose co-transport probably is 1:1 in the case of pars convoluta and 2:1 in membrane vesicles from pars recta. The Na+/L-phenylalanine stoichiometry of the pars convoluta transporter probably is 1:1. Both the high-affinity and the low-affinity Na+-dependent L-phenylalanine transport system of pars recta membrane vesicles seem to operate with a 1:1 stoichiometry. The physiological importance of the arrangement of low-affinity and high-affinity transport systems along the kidney proximal tubule is discussed.

The mechanism of decreased Na +-dependent d-glucose transport in brush-border membrane vesicles from rabbit kidneys with experimental Fanconi syndrome

In our previous paper (Yanase, M. et al. (1983) Biochim. Biophys. Acta 733, 95–101) we reported that the Na +-dependent d-glucose uptake into brush-border membrane vesicles is decreased in rabbits with experimental Fanconi syndrome (induced by anhydro-4-epitetracycline). In the present paper we investigate the mechanism underlying this decrease. d-Glucose is taken up into the osmotically active space in anhydro-4-epitetracycline-treated brush-border membrane vesicles and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as in control brush-border membrane vesicles. The passive permeability properties of control and anhydro-4-epitetracycline-treated brush-border membrane vesicles are shown to be the same as measured by the time-dependence of l-glucose efflux from brush-border membrane vesicles. d-Glucose flux was measured by the equilibrium exchange procedure at constant external and internal Na + concentrations and zero potential. Kinetic analyses of Na +-dependent d-glucose flux indicate that V max in anhydro-4-epitetracycline-treated brush-border membrane vesicles (79.3 ± 7.6 nmol/min per mg protein) is significantly smaller than in control brush-border membrane vesicles (141.3 ± 9.9 nmol/min per mg protein), while the K m values in the two cases are not different from each other (22.3 ± 0.9 and 27.4 ± 1.8 nM, respectively). These results suggest that Na +-dependent d-glucose carriers per se are affected by anhydro-4-epitetracycline, and that this disorder is an important underlying mechanism in the decreased Na +-dependent d-glucose uptake into anhydro-4-epitetracycline-treated brush-border membrane vesicles.

Renal adaptation to phosphate load in the acutely thyroparathyroidectomized rat: rapid alteration in brush border membrane phosphate transport

The sustained in vivo infusion of phosphate into thyroparathyroidectomized rats resulted, after 1 h, in a marked decrease in net phosphate reabsorption, even though the plasma concentration of phosphate continued to rise. This response to phosphate infusion was expressed at the level of the proximal tubule brush border membrane. Within 40 min of the initiation of the infusion the Na+-dependent phosphate uptake system in isolated membrane vesicles was decreased. Phosphate uptake in the absence of Na+, Na+-dependent D-glucose uptake, and 22Na+ uptake were not affected. These findings demonstrate the locus of this parathyroid hormone-independent adaptation and indicate the rapidity with which the membrane transport system is regulated.

Effect of phloretin on na +-dependent d-glucose uptake by intestinal brush border membrane vesicles

In the present work we studied the effects of phloretin on Na +-dependent d-glucose uptake by brush border membrane vesicles isolated from rabbit small intestine. (1) Phloretin had no inhibitory effect on Na +-dependent d-glucose uptake in the presence of equilibrated Na +, although it inhibited the process in the presence of a Na + gradient. Phlorizin inhibited Na +-dependent d-glucose uptake both in the presence and in the absence of a Na + gradient. Phloretin and phlorizin competed with each other for the inhibition of Na +energized d-glucose uptake. These results indicate that phloretin has no direct interaction with the Na +-dependent d-glucose transporter per se, though phloretin and phlorizin may have a common step(s) in their inhibition mechanisms. (2) Phloretin, but not phlorizin, was found to increase the equilibrium level of d-glucose uptake whether Na + was present or not. The increase in the equilibrium level was due to phloretin-induced binding of d-glucose to the brush border membrane, but not due to an increase in the intravesicular space. It seems to occur by a mechanism different from that by which phloretin inhibits Na +energized d-glucose uptake, because the equilibrium level of d-glucose uptake is increased by the ionized form of phloretin but Na +-energized d-glucose uptake is inhibited by the unionized form.

Effects of ethanol in vitro on rat intestinal brush-border membranes

Ethanol, at concentrations found in the intestinal lumen after moderate drinking, has been shown to inhibit carrier-mediated intestinal transport processes. This inhibition could occur by direct interaction with membrane transporters, dissipation of the energy producing Na + electrochemical gradient and/or nonspecific alteration of membrane integrity. The latter alteration may be reflected by changes in membrane fluidity, chemical composition or vesicular size. These possibilities were examined with studies in purified brush border membrane vesicles of rat intestine. Ethanol inhibited concentrative Na +-dependent d-glucose uptake in a dose-dependent manner. In contrast, ethanol did not inhibit concentrative d-glucose uptake under conditions of d-glucose trans-stimulation in the absence of a Na + electrochemical gradient. Ethanol also inhibited initial, concentrative Na +-dependent taurocholic acid uptake, as well as equilibrium uptake. That ethanol exerted a dual effect on transport by increasing membrane conductance for Na + while decreasing intravesicular space was supported by direct studies of Na + uptake. Morphometric analysis confirmed that ethanol-treated membranes had a decreased intravesicular size when compared to untreated membranes. Finally, membrane fluidity measured by EPR showed that ethanol had a significant fluidizing effect without producing qualitative changes in membrane proteins, as determined by SDS gel electrophoresis. These results suggest that ethanol inhibits carrier-mediated transport by dissipation of the Na + electrochemical gradient and alteration of membrane integrity rather than by direct interaction with membrane transporters.

Differential response of enterocytes to vitamin D during embryonic development: induction of intestinal inorganic phosphate, D-glucose and calcium uptake.

Chick intestine maintained in organ culture at day 20 of embryonic life responds to appropriate dose levels of 1,25-dihydroxyvitamin D3 or vitamin D3 in the culture medium by stimulation of Na+-dependent uptake of inorganic phosphate (Pj) and D-glucose as well as by increased calcium absorption. The intensity of each single response to vitamin D varies along the small intestine: Only the jejunum shows high vitamin D increments of all three transport systems under investigation. Inhibition of vitamin D responses by actinomycin D suggests the involvement of gene activation in the sterol's action of raising intestinal transport activities. Determination of the developmental pattern of each vitamin D-dependent transport system showed that only calcium absorption could be induced in undifferentiated cells on day 15, while complete expression of vitamin D action, including elevation of Na+-dependent uptake of D-glucose and Pj, required a fairly advanced state of epithelial maturation and was thus not observed before the final day 20 of embryonic development. This indicates a progressive change of hormone sensitivity of absorptive cells during differentiation.

4-Azidophlorizin, a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes

A new phlorizin derivative ( 2′-O-(β- d- glucopyranosyl)-4- azidophloretin , 4-azidophlorizin) has been synthesized and its affinity for the d-glucose, Na + co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) K′ i, the constant for fully-competitive inhibition of (Na +, Δψ)-dependent d-glucose uptake, (2) K′ d, the dissociation constant of 4-azido[ 3H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) K″ i, the constant for fully-competitive inhibition of the specific ((Na +, Δψ)-dependent, d-glucose protectable) high-affinity [ 3H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants ( K′ i, K′ d and K″ i) were essentially equal and ranged between 3.2 and 5.2 μM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its K′ i values are some 15–20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244–248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.

Phospholipid methylation of kidney cortex brush border membranes. Effect on fluidity and transport

Exposure of intact brush border membrane vesicles of hog kidney cortex to cholesterol oxidase resulted in 24% oxidation of membrane cholesterol compared with more than 95% oxidation of cholesterol in lipids isolated from membranes, showing that cholesterol is asymmetrically distributed in membranes. Phospholipase C, hydrolyzed 76% of phosphatidylcholine and 10–12% phosphatidylethanolamine while phosphatidylserine was not hydrolyzed, thus indicating that majority of phosphatidylcholine is present on the outer surface of these vesicles while phosphatidylethanolamine and phosphatidylserine are present on the inner surface. Methylation of phospholipids in brush border membrane with S- adenosyl-[methyl- 3 H] methionine resulted in the formation of phosphatidyl-N- monomethylethanolamine , phosphatidyl-N,N- dimethylethanolamine and phosphatidylcholine from endogenous phosphatidylethanolamine. The K m for S- adenosylmethionine was 1·10 −4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg 2+ was without any effect between pH 5 to 10. Addition of exogenous mono- and dimethylphosphatidylethanolamine derivatives enhanced methyl group incorporation by 4–5-fold as compared to the addition of phosphatidylethanolamine. The conversion of endogenous phosphatidylethanolamine to phosphatidyl-N- monomethylethanolamine or addition of exogenous phosphatidylmonomethylethanolamine to brush border membrane did not result in a change in bulk membrane fluidity as determined by fluorescence polarization of diphenylhexatriene. Methylation of phosphatidylethanolamine in brush border membrane did not affect the Na +-dependent uptake of either d-glucose or phosphate, although the accessibility of cholesterol in membrane to cholesterol oxidase was diminished by 21%, presumably due to altered flip-flop movement of cholesterol in the membrane.

Interactions between Na +-dependent uptake of d-glucose, phosphate and l-alanine in rat renal brush border membrane vesicles

d-Glucose decreases phosphate reabsorption in rat proximal tubule. It is also postulated that some amino acids interact with phosphate reabsorption. To investigate the mechanism of these interactions, phosphate, d-glucose and l-alanine transport kinetics were measured in brush border membrane vesicles isolated from superficial rat kidney cortex by the calcium precipitation technique. At pH 7.4, Na +-dependent phosphate transport was inhibited in the presence of either d-glucose (39 mM) or l-alanine (2.4 mM). In this model, with d-glucose or with l-alanine the V value of the phosphate uptake was decreased, whereas the apparent K m for the phosphate uptake was not affected. However, some inhibition of phosphate transport was observed in the presence of l-glucose, d-alanine or d-glucose after phlorizin preincubation. A 30% Na +-dependent l-alanine (0.1 mM) transport inhibition was observed in the presence of 5 mM phosphate. d-Glucose (1 mM) was also inhibited by 20% when 5 mM phosphate was added to incubation medium. According to several authors, in our model, d-glucose decreased the l-alanine transport and vice versa. Moreover, when the membrane potential was abolished, a clear inhibition of d-glucose by l-alanine persisted. These multiple interactions could be explained by the accelerated dissipation of the Na + gradient insofar as the rate of the Na + uptake was increased with d-glucose, l-alanine or phosphate and since the absence of variations in membrane potential did not suppress these inhibitions.

Kinetics of d-glucose transport into renal membrane vesicles: Measurement using a vacuum manifold apparatus

The rapid kinetics of d-glucose uptake into membrane vesicles, prepared from the renal cortex of the rat, were measured. A vacuum manifold apparatus for the filtration of suspensions containing membrane vesicles and radiolabelled sugars was constructed to permit measurements of d-glucose accumulation within the vesicles at 8-s intervals. The rate of Na +-independent accumulation of d-glucose was nearly constant for the first 24 s while the rate for Na +-dependent uptake was always changing. While a linear relationship between Na +-dependent d-glucose accumulation and time could not be established for a time period as short as 8 s, the time for half maximum Na +-dependent d-glucose uptake could be estimated. A value of 4 s for half maximum accumulation d-glucose into membrane vesicles in the presence of sodium was obtained.