Action of 5-thio-D-glucose and its 1-phosphate with hexokinase and phosphoglucomutase.

Previous work from this laboratory has shown that 5-thio-d-glucose is a competitive inhibitor for active transport of d-glucose. The present work indicates that the thiosugar analog and its 1-phosphate can also interfere with d-glucose 6-P formation.5-Thio-d-glucose serves as a substrate for yeast hexokinase with a K_m of 4 mm, and V of 8.8 nmol/min/μg of protein. The analog competitively inhibits d-glucose phosphorylation with a K_i of 20 mm.5-Thio-d-glucose 1-P can act as a substrate for rabbit skeletal muscle phosphoglucomutase with a K_m of 60 μm and V of 0.17 μmol/min/μg of protein. Thus, 5-thio-d-glucose 1-P behaves as a near metabolic analog of d-glucose 1-P. 5-Thio-d-glucose 1-P is a competitive inhibitor of d-glucose 1-P conversion to the 6-P with a K_i of 16.2 μm.5-Thio-d-glucose 6-P produced by phosphorylation of 5-thio-d-glucose and by conversion from 5-thio-d-glucose 1-P was identified by chromatographic mobility and by color reactions.     

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 $\text{V}$ value of the phosphate uptake was decreased, whereas the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ 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.     

The effects of oestradiol on nucleoside transport in rat uterus

1. By using the non-metabolized cytidine analogue, cytosine arabinoside, it was possible to examine the mechanism of nucleoside transport in the immature rat uterus in the absence of intracellular utilization of the permeant. It was demonstrated that the uptake of cytosine arabinoside is not accumulative and that it can be competitively inhibited by the addition of a second nucleoside, uridine. Introduction of a concentration gradient of uridine from the medium towards the intracellular water promotes the counterflow of cytosine arabinoside out of the cells against its concentration gradient. These properties indicate that a facilitated-diffusion system is involved in nucleoside transport in the uterus. Further counterflow studies have shown that the transport system has a broad specificity for purine and pyrimidine nucleosides and that it is distinct from the processes that mediate the uptake of sugars, amino acids and purine and pyrimidine bases. 2. Oestradiol injection has no effect on the initial rate of cytosine arabinoside uptake in vitro. The increased amount of the analogue taken up per uterus is simply due to the expansion of the uterine volume that accompanies oestrogen action. 3. It is concluded that the striking increase in uridine uptake, observed in vivo in uteri from oestrogen-treated rats, does not result from an increase in the initial rate of nucleoside transport into the intracellular space of the tissue.     

Transport of d-glucose by brush border membranes isolated from the renal cortex

The transport of d-glucose by brush border membranes isolated from the rabbit renal cortex was studied. At concentrations less than 2 mM, the rate of d-glucose uptake increased linearly with the concentration of the sugar. No evidence was found for a “high-affinity” (μM) saturable site. Saturation was indicated at concentrations of d-glucose greater than 5 mM. The uptake of d-glucose was stereospecific and selectively inhibited by d-galactose and other sugars. Phlorizin inhibited the uptake of d-glucose in the presence and absence of Na⁺. The glycoside was a potent inhibitor of the efflux of d-glucose. Preloading the brush border membrane vesicles with d-glucose, but not with l-glucose, accelerated exchange diffusion of d-glucose. These results demonstrate that the uptake of d-glucose by renal brush borders represents transport into an intravesicular space rather than solely binding. The rate of d-glucose uptake was increased when the Na⁺ in the extravesicular medium was high and the membranes were preloaded with a Na⁺-free medium. The rate of d-glucose uptake was inhibited by preloading the brush border membranes with Na⁺. These results are consistent with the Na⁺ gradient hypothesis for d-glucose transport in the kidney. Thus, the presence of a Na⁺-dependent facilitated transport of d-glucose in isolated renal brush border membranes is indicated. This finding is consistent with what is known of the transport of the sugar in more physiologically intact preparations and suggests that the membranes serve as an effective model system in examining the mechanism of d-glucose transport in the kidney.     

Acidic amino acid transport in animal cells and tissues

1. The occurrence and characterization of acidic amino acid transport in the plasma membrane of a variety of cells and tissues of a number of organisms is reviewed. 2. Several cell types, especially in brain, possess both high- and low-affinity transport systems for acidic amino acids. 3. High-affinity systems in brain may function to remove neurotransmitter amino acid from the extracellular environment. 4. Many cell systems for acidic amino acid transport are energized by an inwardly directed Na+ gradient. Moreover, certain cell types, such as rat brain neurons, human placental trophoblast and rabbit and rat kidney cortex epithelium, respond to an outwardly directed K+ gradient as an additional source of energization. This simultaneous action may account for the high accumulation ratios seen with acidic amino acids. 5. Rabbit kidney has been found to have a glutamate-H+ co-transport system which is subject to stimulation by protons in the medium. 6. Acidic amino acid transport in rat brain neurons occurs with a stoichiometric coupling of 1 mol of amino acid to 2 mol of Na+. For rabbit intestine, one Na+ is predicted to migrate for each mol of amino acid. 7. Uptake in rat kidney cortex and in high-K+ dog erythrocytes is electrogenic. However, uptake in rabbit and newt kidney and in rat and rabbit intestine is electroneutral. 8. Na+-independent acidic amino acid transport systems have been described in the mouse lymphocyte, the human fibroblast, the mouse Ehrlich cell and in rat hepatoma cells. 9. In a number of cell systems, D-acidic amino acids have substantial affinity for transport; D-glutamate, in a number of systems, however, appears to have little reactivity. 10. Acidic amino acid transport in some cell systems appears to occur via the "classical" routes (Christensen, Adv. Enzymol. Relat. Areas Mol. Biol. 49, 41-101, 1979). For example, uptake in the Ehrlich cell is partitioned between the Na+-dependent A system (which transports a wide spectrum of neutral amino acids), the Na+-dependent ASC system (which transports alanine, serine, threonine, homoserine, etc.), and the Na+-independent L system (which shows reactivity centering around neutral amino acids such as leucine and phenylalanine). Also, a minor component of uptake in mouse lymphocytes occurs by a route resembling the A system. 11. Human fibroblasts possess a Na+-independent adaptive transport system for cystine and glutamate that is enhanced in activity by cystine starvation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glucose transport into spinach chloroplasts

The uptake of radioactively labeled hexoses and pentoses into the sorbitol-impermeable (3)H(2)O space (the space surrounded by the inner envelope membrane) of spinach (Spinacia oleracea L.) chloroplasts has been studied using silicone layer filtering centrifugation. Of the compounds tested, d-xylose, d-mannose, l-arabinose, and d-glucose are transported most rapidly, followed by d-fructose and l-arabinose. The rate of l-glucose uptake is only about 5% of that of d-glucose.The transport of d-glucose and d-fructose shows saturation characteristics, the K(m) for d-glucose was found to be about 20 mm. All sugars transport and phloretin inhibit d-glucose transport. The temperature dependency of d-glucose transport appears to have an activation energy of 17 kcal/mol.With low external concentrations of d-glucose the transport into the chloroplasts proceeds until nearly the external concentration is reached inside the chloroplasts.d-glucose transport is inhibited by high d-glucose concentrations in the medium. It is concluded that d-glucose and other hexoses are transported by carrier-mediated diffusion across the inner envelope membrane. This transport is similar to the transport of d-glucose into erythrocytes.     

Reconstitution into liposomes of the B degrees -like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na+ dependent [3H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K+. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [3H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na+ and internal K+. The transporter showed low if there is any tolerance towards the substitution of Na+ or K+ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na+ concentration cooperativity index close to 1 was derived, indicating that 1 Na+ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na+ transport. Optimal rate of 0.1 mM [3H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [3H]glutamine (and [3H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl2, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue alpha-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na+ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B degrees-like.     

Distinction of three types of D-glucose transport systems in animal cells

Immunoblotting of plasma membrane fractions from rat kidney cortex with antibody to human erythrocyte glucose transporter showed a single major cross-reacting material of 48K in basolateral membrane fractions possessing a facilitated diffusion system for D-glucose, but not in brush border membrane fractions which have a Na-dependent active transport system. Cytochalasin B inhibited D-glucose uptake in basolateral membrane vesicles but not in brush border vesicles. Cross-reacting materials of 44-55K were detected in several animal cells exhibiting facilitated diffusion systems, including a hormone dependent system. These results indicate molecular difference between glucose transporters of facilitated diffusion systems and active transport systems.     

Amino acid-dependent increase in hepatic system N activity is linked to cell swelling

Treatment of cultured rat hepatocytes with certain amino acids stimulates the activity of the System N transporter. The present report investigates the mechanism by which the stimulatory amino acids elicit their effect. Activation of System N-mediated transport by amino acids is rapid, cycloheximide-insensitive, and involves neither trans-stimulation nor recruitment of additional carriers to the plasma membrane. In addition, the activation is Na(+)-dependent, supporting the related observation that the most effective stimulatory amino acids are substrates of Na(+)-dependent transport Systems A, ASC, and N whereas substrates of Na(+)-independent System L and non-amino acid metabolites are ineffective. The data suggest that active accumulation of amino acids via Na(+)-dependent carriers is necessary for the activation to occur. The amino acid-dependent stimulation is blocked in a concentration-dependent manner by increasing extracellular K+. Treatment of hepatocytes with an amino acid such as asparagine causes cell swelling and stimulation of System N activity; both of these effects are reduced by hypertonic media. Furthermore, swelling of rat hepatocytes with hypotonic media mimics the System N-stimulatory effects of asparagine. Among the Na(+)-dependent amino acid transport systems present in rat hepatocytes, System N is stimulated preferentially by amino acid-containing or hypotonic media. Collectively, these results demonstrate that cell swelling is a prerequisite for the amino acid-dependent activation of the hepatic System N transporter.     

Substrate specificity of sugar transport by rabbit SGLT1: single-molecule atomic force microscopy versus transport studies

In the apical membrane of epithelial cells from the small intestine and the kidney, the high-affinity Na+/d-glucose cotransporter SGLT1 plays a crucial role in selective sugar absorption and reabsorption. How sugars are selected at the molecular level is, however, poorly understood. Here atomic force microscopy (AFM) was employed to investigate the substrate specificity of rbSGLT1 on the single-molecule level, while competitive-uptake assays with isotope-labeled sugars were performed in the study of the stereospecificity of the overall transport. rbSGLT1-transfected Chinese hamster ovary (CHO) cells were used for both approaches. Evidence of binding of d-glucose to the extracellular surface of rbSGLT1 could be obtained using AFM tips carrying 1-thio-d-glucose coupled at the C1 position to a PEG linker via a vinylsulfon group. Competition experiments with monosaccharides in solution revealed the following selectivity ranking of binding: 2-deoxy-d-glucose >or= 6-deoxy-d-glucose > d-glucose > d-galactose >or= alpha-methyl glucoside; 3-deoxy-d-glucose, d-xylose, and l-glucose did not measurably affect binding. These results were different from those of competitive alpha-methyl glucoside transport assays, where the ranking of inhibition was as follows: d-glucose > d-galactose > 6-deoxy-d-glucose; no uptake inhibition by d-xylose, 3-deoxy-d-glucose, 2-deoxy-d-glucose, or l-glucose was observed. Taken together, these results suggest that the substrate specificity of SGLT1 is determined by different recognition sites: one possibly located at the surface of the transporter and others located close to or within the translocation pathway.     

d-Glucose Transport System of Zymomonas mobilis

The properties of the d-glucose transport system of Zymomonas mobilis were determined by measuring the uptake of nonmetabolizable analogs (2-deoxy-d-glucose and d-xylose) by wild-type cells and the uptake of d-glucose itself by a mutant lacking glucokinase. d-Glucose was transported by a constitutive, stereospecific, carrier-mediated facilitated diffusion system, whereby its intracellular concentration quickly reached a plateau close to but not above the external concentration. d-Xylose was transported by the d-glucose system, as evidenced by inhibition of its uptake by d-glucose. d-Fructose was not an efficient competitive inhibitor of d-glucose uptake, indicating that it has a low affinity for the d-glucose transport system. The apparent K(m) of d-glucose transport was in the range of 5 to 15 mM, with a V(max) of 200 to 300 nmol min mg of protein. The K(m) of Z. mobilis glucokinase (0.25 to 0.4 mM) was 1 order of magnitude lower than the K(m) for d-glucose transport, although the V(max) values for transport and phosphorylation were similar. Thus, glucose transport cannot be expected to be rate limiting at concentrations of extracellular glucose normally used in fermentation processes, which greatly exceed the K(m) for the transport system. The low-affinity, high-velocity, nonconcentrative system for d-glucose transport described here is consistent with the natural occurrence of Z. mobilis in high-sugar environments and with the capacity of Z. mobilis for rapid conversion of glucose to metabolic products with low energetic yield.     

Transport and metabolism of galactose in rat kidney cortex

1. Analysis of transport of d-galactose was complicated by metabolism of the compound but appeared to have two components: a substrate-saturable component and a diffusion component. At low substrate concentration (<1mm) active transport was observed. Accumulation of galactose was largely independent of Na(+) concentration. The apparent K(m) for this component was 0.2mm. At substrate concentrations above 1mm the active transport system appeared saturated and further increases in substrate concentration resulted in a linear increase in the rate of galactose accumulation, but no concentration gradient was formed. 2. d-[1-(14)C]Galactose (2mm) was metabolized to (14)CO(2) by rat kidney-cortex slices incubated at 37 degrees C, at the rate of 68nmol/h per 100mg of tissue. 3. Intracellular components from such incubations were separated into a neutral fraction, the only major labelled component being galactose, and a phosphorylated fraction. 4. Phosphorylated metabolites found in galactose-incubated slices increased with increasing substrate concentration and achieved a limiting value of 0.42mm after 60min of incubation. 5. Galactose uptake was inhibited by anaerobiosis, dinitrophenol and phlorrhizin. 6. Methyl alpha-d-glucoside and d-glucose partially inhibited galactose uptake only at ratios of 100:1. 7. The presence of pyruvate did not decrease galactose metabolism although it did decrease production of (14)CO(2) from [1-(14)C]galactose. Gluconeogenesis occurred in the presence of pyruvate and (14)C from galactose was found in glucose. 8. Rat kidney-cortex slices metabolized 2mm-[1-(14)C]galactonate to (14)CO(2) at a rate of 20nmol/h per 100mg of tissue.     

A study of the sulphur amino acids of rat tissues

1. In a study of the metabolism of l-[(35)S]methionine in vivo, the labelled sulphur compounds of rat liver and brain were separated first by ion-exchange chromatography into two fractions containing (i) free sulphur amino acids such as methionine, cystathionine, cyst(e)ine and homocyst(e)ine and (ii) glutathione. 2. Two-dimensional paper chromatography with butan-1-ol-acetic acid or propionic acid-water in the first direction and 80% acetone or acetone-ethyl methyl ketone-water in the second direction was found superior to other solvent systems for separating the sulphur amino acids. 3. At 10min. after injection of [(35)S]methionine only a small part of the (35)S was found combined in free methionine or other free sulphur amino acids. 4. Evidence was obtained of the presence of adenosyl[(35)S]methionine and adenosyl[(35)S]homocysteine in perchloric acid extracts of rat liver and brain. 5. The trans-sulphuration pathway was active in brain as well as in liver.     

Transport of L-tryptophan into slices of rat cerebral cortex

—Slices of rat cerebral cortex, when incubated aerobically at 37°C in Krebs-Ringer-phosphate solution (pH 7.0) containing 10 mm glucose and 1.0 mm l -tryptophan [1-¹⁴C], accumulated tryptophan. Within the first 15 min of incubation the ratio of the concentration of the amino acid in the tissue to that in the medium reached 3.5:1. Uptake of tryptophan was linear for the first 30 min and attained a maximum concentration ratio (tissue:medium) of 6.5:1 within 60 min. The transport mechanism became saturated at 1.0-3.0 mm tryptophan. Entry of the amino acid into the cortical cells was thereafter directly proportional to its initial concentration in the medium. The tissue: medium ratio at 15 min decreased significantly under the following experimental conditions: (1) lowering the incubation temperature to 0°C; (2) incubating under N₂; (3) omitting glucose; (4) decreasing the Na⁺ concentration below 50 mm ; (5) removing K⁺ from the medium; or (6) adding 1.0 mm NaCN or 0.1 mm protoveratrine B to the medium. These results provided evidence that the accumulation of tryptophan against its concentration gradient was an active process. The effects of a number of amino acids on the uptake of tryptophan were studied: of these, l -phenylalanine, dl -p-chlorophenylalanine, l -tyrosine, l -DOPA, the branched chain aliphatic amino acids (l -leucine, l -isoleucine, l -valine) and l -glutamic acid were found to be the most potent inhibitors of tryptophan transport. Several tryptophan metabolites were tested; only l -kynurenine inhibited the uptake of tryptophan.     

On the mechanism of sugar and amino acid interaction in intestinal transport.

The influence of amino acids on D-glucose transport was studied in isolated vesicles of brush border membrane from rat small intestine. It is demonstrated that: (a) Uptake of D-glucose by the membranes is inhibited by simultaneous flow of L- and D-alanine into the vesicles. (b) Addition of L-alanine to membranes pre-equilibrated with D-glucose causes efflux of this sugar. (c) The influence of amino acids on D-glucose is dependent on the presence of Na+. (d) The ionophorous agents monactin and valinomycin are able to prevent the transport interaction of D-glucose and amino acids. Monactin is effective in the presence of Na+ without further addition of other cations, while valinomycin is effective only with added K+, in accordance with the known specificity of these antibiotics. (e) The inhibitory effect increases with L-alanine concentration up to about 50 mM after which it levels off. The experiments provide evident that the Na+-dependent sugar and amino acid fluxes across the brush border membrane are coupled electrically.     

Interaction of the trp repressor from Escherichia coli with a constitutive trp operator.

The mechanisms of the requirement of glucose for steroidogenesis were investigated by monitoring the uptake of the glucose analogue 2-deoxy-D-glucose by rat testis and tumour Leydig cells. The characteristics of glucose transport in both of these cell types were found to resemble those of the facilitated-diffusion systems for glucose found in most other mammalian cells. The Leydig cells took up 2-deoxy-D-glucose but not L-glucose, and the uptake was inhibited by both cytochalasin B and forskolin. In the presence of luteinizing hormone, the rate of 2-deoxy-D-glucose uptake by both cell types was increased by approx. 50%. In addition to D-glucose, it was shown that the Leydig cells could also utilize 3-hydroxybutyrate or glutamine to maintain steroidogenesis.     

gamma-Glutamyl amino acids. Transport and conversion to 5-oxoproline in the kidney

Transport of gamma-glutamyl amino acids, a step in the proposed glutathione-gamma-glutamyl transpeptidase-mediated amino acid transport pathway, was examined in mouse kidney. The transport of gamma-glutamyl amino acids was demonstrated in vitro in studies on kidney slices. Transport was followed by measuring uptake of 35S after incubation of the slices in media containing gamma-glutamyl methionine [35S]sulfone. The experimental complication associated with extracellular conversion of the gamma-glutamyl amino acid to amino acid and uptake of the latter by slices was overcome by using 5-oxoproline formation (catalyzed by intracellular gamma-glutamyl-cyclotransferase) as an indicator of gamma-glutamyl amino acid transport. This method was also successfully applied to studies on transport of gamma-glutamyl amino acids in vivo. Transport of gamma-glutamyl amino acids in vitro and in vivo is inhibited by several inhibitors of gamma-glutamyl transpeptidase and also by high extracellular levels of glutathione. This seems to explain urinary excretion of gamma-glutamylcystine by humans with gamma-glutamyl transpeptidase deficiency and by mice treated with inhibitors of this enzyme. Mice depleted of glutathione by treatment with buthionine sulfoximine (which inhibits glutathione synthesis) or by treatment with 2,6-dimethyl-2,5-heptadiene-4-one (which effectively interacts with tissue glutathione) exhibited significantly less transport of gamma-glutamyl amino acids than did untreated controls. The findings suggest that intracellular glutathione functions in transport of gamma-glutamyl amino acids. Evidence was also obtained for transport of gamma-glutamyl gamma-glutamylphenylalanine into kidney slices.     

Site of uptake of nonfiltered amino acid in the rabbit kidney

During passage from renal artery to vein, nonfiltered amino acids are known to be extracted by the kidney, an observation generally attributed to their basolateral uptake by tubular epithelium. This attribution is here tested in the rabbit, using the nonmetabolizable analogue cycloleucine as test compound. Uptake of cycloleucine is diffusion limited and could be maximized by lengthening its artery-to-vein transit time by short aortic occlusion. The transient anoxia did not abolish active solute transport in the kidney; the technique permits acute loading of the kidney with, for instance, a nephrotoxicant without excessive exposure of the whole animal. The major portion of cycloleucine taken up by nonfiltering kidneys during occlusion returned to renal venous plasma with a mean delay of 45 sec, as if it had accumulated in the same cellular transport pool through which reabsorbed cycloleucine has to pass. A fraction of the amino acid taken up also reached the tubular lumen. These results support the suggested role of tubule cells in the extraction of amino acids from postglomerular blood.