Role of -SH groups in rat sugar intestinal transport in vivo.

The effect of p-chloromercuribenzoic acid (pCMB), either alone or in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), on the 1 mM galactose absorption by in vivo perfused rat intestine has been studied. At 0.25 mM concentration, pCMB inhibits galactose absorption in about 32% but it does not modify the absorption of this sugar when the transport is blocked by 0.5 mM phlorizin, or that of the non-transportable monosaccharide derivative 2-deoxy-D-glucose. This shows that only the active transport component of galactose absorption is inhibited. A 2 min preexposure period is required for the inhibition to appear. The inhibition was not reversed by washing with saline solution even when it contained 0.5 mM dithioerythritol, 10 mM cysteine or 5 and 10 mM EDTA. The simultaneous exposure to 0.25 pCMB and 0.25 mM DTNB inhibits the total galactose entry in about 50%, an effect higher than the one exerted by each reagent separately and close to the one obtained with 0.5 mM phlorizin. Our results, in vivo, confirm the importance of the thiol groups in the cotransport of Na+ and sugar. As DTNB is an SH-reagent of lesser liposolubility than pCMB, the existence of two populations of sulfhydryl groups related to sugar transport which differ in their location within the brushborder membrane and in accessibility from the intestinal lumen, is suggested.     

A hydrolase-related transport system is not required to explain the intestinal uptke of glucose liberated from phlorizin

The fate of [3H]glucose released from a wide range of [3H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na+-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2-2.0 mM phlorizin, the [3H]glucose uptake was a constant 11-12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [3H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [14C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [14C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na+-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medum by virtue of the position of the site where it is formed, i.e inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.     

Isoosmotic transport of fluid across the hamster small intestine in the presence of phlorizin-induced inhibition of sugar transport.

Experiments were performed to investigate whether the fluid transported across the small intestine is isoosmotic with the mucosal solution when the active transport of glucose is partially inhibited. Everted hamster mid small intestine was incubated in one of the following four mucosal solutions: (1) Isotonic control, Krebs-Ringer bicarbonate solution containing 10 mM glucose (KRBSG), (2) Isotonic with phlorizin, KRBSG + 5X10-5 M phlorizin, (3) Hypertonic control, KRBSG + 50 mM mannitol, (4) Hypertonic with phlorizin, KRBSG + 50 MM mannitol + 5x10-5 M phlorizin. The serosal surface of the intestine was not bathed. Results indicate that the transported fluid was always isoosmotic with any of the mucosal solutions used. When the mucosal solution was made hypertonic with mannitol, the concentration of glucose and electrolytes in the absorbate increased, and as a result, the absorbate became hypertonic and isoosmotic with the mucosal solution. The presence of phlorizin either in the isotonic or in the hypertonic mucosal solution decreased the glucose concentration of the absorbate, but the transported fluid became isoosmotic with the mucosal solution due to a higher concentration of Na, K, and their associated anions. Phlorizin caused a decrease in the transmural potential difference. In spite of this, the presence of this glucoside in the mucosal solution increased the transport of sodium in relation to glucose transport. It is suggested that, at the concentrations used, phlorizin inhibits sodium movement through the electrogenic pathway, but increases the transport of this ion through the non-electrogenic route. This increase in neutral sodium transport seems to compensate for the low concentration of glucose in the absorbate, so that the absorbate becomes isoosmotic with the mucosal solution whether the latter is isotonic or hypertonic. It is suggested further that isoosmotic transport of fluid is an inherent property of the small intestine and that there may be an osmoregulatory mechanism in the gut which controls this process.     

Uranyl action on sugar transport across rat jejunum

The effect of uranyl on sugar transport across rat jejunum has been studied in vitro and in vivo. D-glucose and D-galactose accumulation in jejunum rings at pH 6.0 is inhibited about 40-65% by 1 mM uranyl nitrate. This inhibition is lower than that produced by 0.5 mM phlorizin. The effect was very similar when the incubation of the rings with the sugar was made in the absence of uranyl, after preincubation with the inhibitor. Washing with 10 mM EDTA reverted uranyl inhibition only slightly. D-fructose entry was weakly inhibited by uranyl. Glucose absorption in vivo along perfusion periods of 1 min was not affected by the presence of uranyl (0.001 to 1 mM) in the sugar solution, but the exposure of the mucosa to 0.1 mM uranyl at pH 6.5 for 10 min inhibited sugar absorption at the same pH in the subsequent periods of perfusion. Results suggest that uranyl impairs sugar transport by binding to protein chemical groups at the surface or in deeper sites of enterocyte membranes, a process that requires some minutes to be accomplished.     

The Na+ gradient-dependent transport of D-glucose in renal brush border membranes.

The Na+-dependent transport of D-glucose was studied in brush border membrane vesicles isolated from the rabbit renal cortex. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium induced a marked stimulation of D-glucose uptake. Accumulation of the sugar in the vesicles reached a maximum and then decreased, indicating efflux. The final level of uptake of the sugar in the presence of the Na+ gradient was identical with that attained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the overshoot the uptake of D-glucose was more than 10-fold the equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of D-glucose into renal brush border membranes against its concentration gradient. The stimulation of D-glucose uptake into the membranes was specific for Na+. The rate of uptake was enhanced with increased concentration of Na+. Increasing Na+ in the external medium lowered the apparent Km for D-glucose. The Na+ gradient effect on D-glucose transport was dissected into a stimulatory effect when Na+ and sugar were on the same side of the membrane (cis stimulation) and an inhibitory effect when Na+ and sugar were on opposite sides of the membrane (trans inhibition). The uptake of D-glucose, at a given concentration of sugar, reflected the sum of the contributions from a Na+-dependent transport system and a Na+-independent system. The relative stimulation of D-glucose uptake by Na+ decreased as the sugar concentration increased. It is suggested, however, that at physiological concentrations of D-glucose the asymmetry of Na+ across the brush border membrane might fully account for uphill D-glucose transport. The physiological significance of the findings is enhanced additionally by observations that the Na+-dependent D-glucose transport system in the membranes in vitro possessed the sugar specificities and higg phlorizin sensitivity characteristic of more intact preparations. These results provide strong experimental evidence for the role of Na+ in transporting D-glucose across the renal proximal tubule luminal membrane.     

Kinetics of uptake of glucose in rabbit jejunum: influence of sodium, unstirred layers, and passive permeation

Failure to account for the effect of the unstirred water layer and the contribution of passive permeation will lead to errors in the estimation of the kinetic constants of glucose uptake into the intestine. It is widely accepted that variations in the concentration of sodium in the bulk phase profoundly influence the rate of uptake of glucose in the intestine, but the kinetic basis for this effect remains in dispute. Accordingly, a previously validated in vitro technique was used to assess the effect of Na+ on the uptake of glucose into rabbit jejunum under conditions selected to reduce the unstirred layer resistance. Varying Na+ had no effect on the uptake of lauryl alcohol and therefore on unstirred layer resistance. The passive permeability coefficient for glucose uptake was estimated from the uptake of L-glucose, of D-glucose at 4 degrees C, or in the presence of 1 mM phlorizin or 40 mM galactose. The permeability for glucose increased as Na+ rose. The values of both the maximal transport rate and the Michaelis constant (Km) were influenced by Na+. A linear relationship was noted between Na+ and the maximal transport rate; the value of Km fell as Na+ was increased to 75 mequiv./L, but Km did not decline further with higher values of Na+. These results support the theoretical predictions of the presence of both an affinity and a velocity effect of the sodium gradient on the intestinal transport system for glucose.     

Inhibition of sugar active transport across rat intestine in vivo by cadmium

Galactose absorption by rat jejunum perfused in vivo is inhibited by 0.5 mM Cd2+. This effect is explained by impairment of the phlorizin-sensitive sugar transport system, as Cd does not modify the absorption of L-sorbose or that of galactose in the presence of 0.5 mM phlorizin. Cd inhibition is observed as early as in the 1st minute, does not increase by previous exposure of the mucosa to the metal and does not decrease after washing with saline solution, but it is entirely reversed by EDTA or dithioerythritol. Results agree with a Cd2+ binding to HS- groups of membrane proteins at the brush border, appertaining or functionally related to the phlorizin-sensitive and Na+ dependent transport system for sugars.     

Transport and phosphorylation of D-galactose in renal cortical cells.

An improved analytical procedure for the extraction and determination of total, free and phosphorylated tissue sugar is described. This method, employing ZnSO4 plus Ba(OH)2 for the precipitation of sugar phosphates, yields values identical with those obtained by the more laborious separation of free and phosphorylated sugar by ion-exchange chromatography. Erroneous values for free sugar due to the action of a Zn2+ -activated phosphatase and/or the lability to acids of some sugar phosphates, are avoided. Using this technique for the sudy of transport and phosphorylation of D-galactose in rabbit renal cortical slices and tissue extracts, it was found: 1. The cellular uptake of D-galactose was associated with the appearance of both free and phosphorylated sugar whether or not external Na+ was present. At 1 mM sugar, galactose was accumulated in the cells against a modest concentration gradient of 1.445 +/- 0.097 (n = 17). Galactose phosphate appeared in the cells considerably faster than free sugar under conditions of net uptake as well as of steady-state exchange (pulse-labelling). 2. Increasing saline pH (6-8) increased the cellular levels of sugar phosphate without affecting the steady-state values of free sugar. With tissue extracts, increasing pH also stimulated the activity of galactokinase and the dephosphorylation of galactose 1-phosphate by a Zn2+ -activated phosphatase. 3. 0.5 mM phlorizin inhibited the tissue uptake of galactose and its subsequent oxidation to CO2 only to a minor degree (30 and 10%, respectively). The absence of external Na+ further depressed the phlorizin effect. Preincubation of the tissue with phlorizin and subsequent washing in part abolished the inhibitory effect. The data suggest that a major portion of the galactose uptake by the tissue proceeds by a mechanism with a low affinity for phlorizin. 4. Efflux studies showed that the wash-out of free galactose from slices was associated with a net decrease of both free and phosphorylated tissue sugar. 5. The above results suggest the possibility that phosphorylation may represent a step in the Na+ -independent, phloretin-sensitive transfer of D-galactose across the antiluminal cell membrane. The participation of intracellular galactokinase and a Zn2+ -activated alkaline phosphatase in the maintenance of the steady state of free and phosphorylated galactose in the cells has been demonstrated.     

Ion Transport in Isolated Rabbit Ileum : II. The interaction between active sodium and active sugar transport

The addition of actively transported sugars to the solution bathing the mucosal surface of an in vitro preparation of distal rabbit ileum results in a rapid increase in the transmural potential difference, the short-circuit current, and the rate of active Na transport from mucosa to serosa. These effects are dependent upon the active transport of the sugar per se and are independent of the metabolic fate of the transported sugar. Furthermore, they are inhibited both by low concentrations of phlorizin in the mucosal solution and by low concentrations of ouabain in the serosal solution. The increase in the short-circuit current, ΔI_sc, requires the presence of Na in the perfusion medium and its magnitude is a linear function of the Na concentration. On the other hand, ΔI_sc is a saturable function of the mucosal sugar concentration which is consistent with Michaelis-Menten kinetics suggesting that the increase in active Na transport is stoichiometrically related to the rate of active sugar transport. An interpretation of these findings in terms of a hypothetical model for intestinal Na and sugar transport is presented.     

32P]ATP synthesis in steady state from [32P]Pi and ADP by Na+/K(+)-ATPase from ox brain and pig kidney. Activation by K+

The ouabain-sensitive synthesis of [32P]ATP from [32P]Pi and ADP (vsyn) was measured in parallel with the ouabain-sensitive hydrolysis of [32P]ATP (vhy) at steady state, at varying concentrations of sodium, potassium, magnesium, inorganic phosphate, ADP, ATP and oligomycin, and at varying pH. Na+ was necessary for ATP synthesis, but vsyn was decreased by high sodium concentrations. Oligomycin, depending on the Na+ concentration, either decreased or did not affect vsyn. Potassium, at low concentrations (1-5 mM) increased vsyn at all magnesium and sodium concentrations tested, lower potassium concentrations being needed to activate vsyn at lower sodium concentrations. vsyn was optimal below pH 6.7, decreasing abruptly at higher values of pH. At pH 6.7, vsyn was a hyperbolic function of the concentration of inorganic phosphate. In the presence of potassium, half-maximal rate was obtained at [Pi] congruent to 40 mM, whereas a higher concentration was needed to obtain half-maximal rate in the absence of K+. In contrast, increasing the concentration of ADP caused a nonhyperbolic activation of vsyn, the pattern obtained in the presence of potassium being different from that obtained in its absence. Increasing the ATP concentration above 0.5 mM decreased vsyn. The data are used to elucidate (1) which reaction steps are involved in the ATP-synthesis catalysed by the Na+/K(+)-ATPase at steady state in the absence of ionic gradients and (2) the mechanism by which K+ ions stimulate the reaction.     

Intracellular and extracellular concentrations of Na+ modulate Mg2+ transport in rat ventricular myocytes

Apparent free cytoplasmic concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) were estimated in rat ventricular myocytes using fluorescent indicators, furaptra (mag-fura-2) for Mg2+ and sodium-binding benzofuran isophthalate for Na+, at 25 degrees C in Ca2+-free conditions. Analysis included corrections for the influence of Na+ on furaptra fluorescence found in vitro and in vivo. The myocytes were loaded with Mg2+ in a solution containing 24 mM Mg2+ either in the presence of 106 mM Na+ plus 1 mM ouabain (Na+ loading) or in the presence of only 1.6 mM Na+ to deplete the cells of Na+ (Na+ depletion). The initial rate of decrease in [Mg2+]i from the Mg2+-loaded cells was estimated in the presence of 140 mM Na+ and 1 mM Mg2+ as an index of the rate of extracellular Na+-dependent Mg2+ efflux. Average [Na+]i, when estimated from sodium-binding benzofuran isophthalate fluorescence in separate experiments, increased from 12 to 31 mM and 47 mM after Na+ loading for 1 and 3 h, respectively, and decreased to approximately 0 mM after 3 h of Na+ depletion. The intracellular Na+ loading significantly reduced the initial rate of decrease in [Mg2+]i, on average, by 40% at 1 h and by 64% at 3 h, suggesting that the Mg2+ efflux was inhibited by intracellular Na+ with 50% inhibition at approximately 40 mM. A reduction of the rate of Mg2+ efflux was also observed when Na+ was introduced into the cells through the amphotericin B-perforated cell membrane (perforated patch-clamp technique) via a patch pipette that contained 130 mM Na+. When the cells were heavily loaded with Na+ with ouabain in combination with intracellular perfusion from the patch pipette containing 130 mM Na+, removal of extracellular Na+ caused an increase in [Mg2+]i, albeit at a very limited rate, which could be interpreted as reversal of the Mg2+ transport, i.e., Mg2+ influx driven by reversed Na+ gradient. Extracellular Na+ dependence of the rate of Mg2+ efflux revealed that the Mg2+ efflux was activated by extracellular Na+ with half-maximal activation at 55 mM. These results contribute to a quantitative characterization of the Na+-Mg2+ exchange in cardiac myocytes.     

Examination of substrate-induced conformational changes in the Na+/glucose cotransporter

Conformations of the Na+/glucose cotransporter were examined using tryptophan fluorescence and substrates to induce cotransporter conformational changes. Addition of Na+ but not K+ or TMA+ resulted in a saturable quenching of tryptophan fluorescence with a K0.5 for Na+ of 28 mM. In the presence of saturating Na+ concentrations, d-glucose but not l-glucose, fructose, or phlorizin resulted in a partial return of tryptophan fluorescence to approximately 70% of the substrate-free levels. This return of tryptophan fluorescence was a saturable function of d-glucose concentration with a K0.5 of 43 microM. The three conformations were compared with respect to their sensitivity to tryptophan quench reagents. Acrylamide quenching was unaffected by substrates. In contrast, I- quenching decreased 40% in the presence of Na+, while Cs+ quenching increased 64%. Addition of saturating d-glucose concentrations resulted in the return of I- quenching to 90% of the substrate-free values and reduced Cs+ quenching to substrate-free levels. In contrast, phlorizin did not mimic the effect of d-glucose on tryptophan fluorescence. These results are interpreted in terms of a second substrate-induced cotransporter conformational change which based on similar substrate specificities appears directly related to cotransporter-mediated Na+ and d-glucose transport.     

D-galactose uptake by snail intestine: competitive inhibition by phlorizin and sodium dependence

The net entry of galactose into the tissue of snail everted intestinal rings with 2 or 15 minute long incubation periods has been measured. With 10(-4) M phlorizin, the mediated transport is completely blocked while only the passive entry of sugar is produced. Lower concentrations of the glycoside partially inhibit transport according to competitive inhibition kinetics (K1 = 10(-7) M). The transport of galactose is Na+ dependent. In the absence of Na+, transport ceases and the sugar entry can be explained through simple diffusion. With 15 mM Na+ (control 71,4 mM) transport diminishes and a marked increase in the apparent Km with no changes in the Vmax is observed. One mM harmaline completely blocks galactose (0.5 mM) transport. One mM ouabain also makes transport null, but only after tissue preincubation with the inhibitor on the serosal side.     

Ion activities in the lateral intercellular spaces of gallbladder epithelium transporting at low external osmolarities

The ion activities in the lateral spaces of the unilateral preparation of the gallbladder of Rana catesbiana were measured by double-barrelled ion-selective microelectrodes. The bladders were bathed in a saline solution with a low osmolarity (62 mOsm) containing, in mM: 27 Na+, 27 Cl-, 2 K+, 1 Ca++, 4 HCO3-. Working at reduced osmolarities had the advantage of an increased volume transport and of widened intercellular spaces. The reference barrel recorded an electrical potential of +2.7 mV in the spaces; they contained a solution similar to the external solution. The electrodes recorded a Na+ concentration of 27 mM, a K+ concentration of 1.7 mM, a Ca++ concentration of 0.69 mM and a Cl- concentration of 28.5 mM. In the spaces there was a lower resistance between the tip of the electrode and the serosal bath than that recorded with the tip in the lumen, and injection of fluorescent dye (11 A diameter) via the electrodes did not stain the cells. The concentrations in the secretion were similar to those in the spaces. The intracellular compartment had an apparent K+ concentration of 95 mM, and the concentrations of Na+ and Cl- were both about 5 mM. These data indicate that when the gallbladder is bathed with hypotonic solutions and is transporting fluid at approximately three or four times the normal rate, there are no significant osmotic gradients between the lumen and the lateral spaces. It is suggested that transcellular transport of water is implemented by a combination of high osmotic permeabilities across both mucosal and serosal cell membranes and low reflection coefficients (for K+ salts) at the serosal cell membranes.     

Copper-sodium linkage during intestinal absorption: inhibition by amiloride.

The possible association between copper and sodium small intestinal absorption in the rat was investigated in the presence or absence of the electrolyte transport inhibitors amiloride, acetazolamide, and furosemide, at pharmacologic concentrations, using an in situ perfusion procedure. Amiloride (1 mM) produced a significant decrease in copper, net water, and sodium absorption, in solutions with sodium. Copper tissue retention was not altered, but was much higher in the absence of sodium. Acetazolamide and furosemide (1 mM), in separate experiments, had no effect on copper removal from the lumen, but generally reduced sodium and water transport. The presence or absence of sodium in the perfusate influenced rates of copper uptake. These data are compatible with a more effective passage of copper across the enterocyte basolateral membrane in the presence of sodium than in its absence.     

Dissimilar effects of Na+ and K+ on the promotion of glucosyltransferase secretion in Streptococcus salivarius

A defined growth medium (designated AP11), in which the concentrations of Na+ and K+ could be altered independently of one another, was developed for Streptococcus salivarius ATCC 25975. The addition of 100 mM-Na+ to AP11-medium containing 25 mM-K+ initially reduced the rate of expression of extracellular glucosyltransferase (GTFe). However, once S. salivarius had adaptated to grow in the presence of 100 mM-Na+, the rate of GTFe expression was stimulated. In fact once adapted to the presence of Na+ in the environment the same increase in the rate of enzyme expression was observed in all batch cultures irrespective of the K+ concentration (2-50 mM). At 2 mM-K+ there was no change in the level of saturation of the membrane lipids when the Na+ concentration was increased from 0 mM to 100 mM. Na+-stimulation of GTFe expression was confirmed in non-proliferating cell suspensions at different K+ concentrations. In non-proliferating cell suspensions, GTFe expression outlined a rectangular hyperbola with respect to K+ concentration when the K+ concentration was stepped up from 2 mM. The increase in GTFe synthesis and secretion was transient and was similar to that previously reported by us in Na+-rich medium, though it did not reach the same high levels. The reduced transient stimulation of GTFe expression correlated both with an enrichment in the saturated fatty acids of the membrane lipids of S. salivarius, and with the fact that the degree of saturation was only slightly reduced when the K+ concentration was stepped up from 2 mM to 50 mM. Needless to say, the final octadecenoic to octadecanoic (C18:1/C18:0) fatty acid ratio retained its direct correlation with the transient increase in GTFe production following the step up in K+ concentration, giving rise to an apparent biphasic plot when combined with that previously reported.     

Phosphorylation from adenosine triphosphate of sodium- and potassium-activated adenosine triphosphatase. Comparison of enzyme-ligand complexes as precursors to the phosphoenzyme.

The relative effectiveness of the ligands Mg2+, Na+, and ATP in preparing sodium plus potassium ion transport adenosine triphosphatase for phosphorylation was studied by means of a rapid mixing apparatus. Addition of 2 mM MgC12, 120 mM NaC1, and 5 muM [gamma-32P]ATP simultaneously to the free enzyme gave an initial phosphorylation rate of about 0.3 mu mol-mg-1-min-1 at 25 degrees and pH7.4. Addition of Mg2+ to the enzyme beforehand, separately or in combination with Na+ or ATP, had little effect on the initial rate. Addition of Na+ only to the enzyme beforehand increased this rate 1.5- to 3-fold. Early addition of ATP 130 ms before Na+ plus Mg2+ increased the rate 6- to 7-fold. Early addition of Na+ plus ATP was most effective; it increased the rate about 10-fold. The data indicate that Na+ and ATP bind in a random order and that each ligand potentiates the effect of the other. The rate of dissociation of ATP from the enzyme was estimated by a chase of unlabeled ATP of variable duration. This rate was slowest in the presence of Mg2+ (k = 540 min-1), most rapid in the presence of Na+ (k = 2000 min-1), and intermediate (k = 1100 min-1) in the absence of metal ions. The effect of Na+ concentration on the rate of phosphorylation was estimated when Na+ with Mg2+ was added to the enzyme-ATP complex. The rate followed Michaelis-Menten kinetics with a maximum of 2.9 mu mol-mg-1 and a Km of 8 mM. The effect of Na+ concentration was also estimated on the increment in the rate of phosphorylation produced by the presence of Na+ with the enzyme-ATP complex beforehand. The increment followed the same kinetics with a maximum of 3.75 mu mol-mg-1-min-1 and a Km of 5.4 mM. In both cases estimation of the Hill coefficient failed to show cooperativity between binding sites for Na+. In contrast, the dependence of ouabain-sensitive ATPase activity on Na+ concentration in the absence of K+ indicated two sites for Na+ with apparent Km values of 0.16 and 8.1 mM, respectively.     

Glucose regulation of pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase

The effect of glucose and 2-deoxy-D-glucose on pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase has been investigated by following pH transients in a stopped-flow spectrophotometer. A typical pre-steady state signal showed an initial decrease then subsequent increase in acidity. Under optimal Na^＋ （120 mM） and K^＋ （30 mM） concentrations, magnitudes of both H^＋ release and H^＋ absorption were found to be approximately 1.0/ATPase molecule. The presence of 1 mM glucose significantly decreased H^＋ absorption at high Na^＋ concentrations, whereas it was ineffective at low Na^＋. H^＋ release was decreased significantly in the presence of 1 mM glucose at Na^＋ concentrations ranging from 30 mM to 120 mM. Similar to the control, K^＋ did not show any effect on either H^＋ release or H^＋ absorption at all tested combinations of Na^＋ and K^＋ concentrations. Pre-steady state H^＋ signal obtained in the presence of 2-deoxy-D-glucose did not vary significantly as compared with glucose. Delayed addition of K^＋ （by 30 ms） to the mixture （enzyme＋ 120 mM Na^＋ATP＋glucose） showed that only small fractions of population absorb H^＋ in the absence of K^＋. No H^＋ absorption was observed in the absence of Na^＋. Delayed mixing of Na^＋ or K^＋ did not have any effect on H^＋ release. Effect of 2-deoxy-D-glucose on H^ absorption and release was almost the same as that of glucose at all combinations of Na^＋ and K^＋ concentrations. Results obtained have been discussed in terms of an extended kinetic scheme which shows that, in the presence of either glucose or 2-deoxy-D-glucose, significantly fewer enzyme molecules reache the E-P（3Na＋） stage and that K^ plays an important role in the conversion of E1 .ADP.P（3Na^＋） to H^＋.E1-（3Na^＋） complex.     

Change in membrane elastic modulus on activation of glucose transport system of brush border membrane vesicles studied by osmotic swelling and dynamic light scattering.

The cell membrane having a transport system is inferred to be flexible when its function is being activated. For the brush border membrane vesicles prepared from rat small intestine, which have the co-transport system of Na+ and glucose, the membrane elasticity was measured as a function of the d-glucose concentration in the presence of Na+ ions. The elastic modulus of the vesicle membrane was obtained by an osmotic swelling method. Osmolality was changed by diluting the extravesicular d-mannitol concentration. The change in the diameter of the membrane vesicle in response to an osmolality change was measured by the dynamic light-scattering method. The elastic modulus of the vesicle membrane decreased from 150 dyn/cm to 80 (45) dyn/cm with the increase of d-glucose, from 0 mM to 10(30) mM in the presence of 10 mM Na+ ions. On the other hand, in the presence of 1 mM phlorizin, a glucose-transport inhibitor, the elastic modulus remained at a constant value of 160 dyn/cm in the same range of the d-glucose concentration. This indicates that the vesicle membrane becomes flexible when its transport function is activated. In a broad osmolality range, the brush border membrane vesicle showed cycles of "swell-burst-reseal". The vesicle membrane became flexible after every cycle, namely, the modulus was 150, 120, and 55 in units of dyn/cm in the presence of 1 mM d-glucose and 50 mM Na+ ions.     

Sodium: a regulator of glucose uptake in virus-transformed and nontransformed cells.

Observations of cells transformed by the Bryan strain of Rous sarcoma virus (RSV-BH) suggested that the intracellular concentrations of sodium ion (Na+) may play a critical role in cellular metabolism. In an attempt to manipulate intracellular Na+, chick embryo cells were exposed to graded concentrations of Na+ in the cellular growth medium, and the effects on capacity for glucose uptake was examined. After incubation for six hours, the incorporation rate of 2-deoxyglucose (used as a substitute for glucose) was proportional to the external Na+ concentration over the range, 100 mM to 200 mM. Cells transformed by RSV-BH were less responsive than nontransformed cells to differences in Na+ at low concentrations. The changes were specifically dependent upon Na+, since K+, Li+, or choline + were ineffective as substitutes, and increasing the ionic strength above that of 120 mM Na+ was effective only when Na+ was the added cation.