Immobilization of d-glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and d-glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized d-glucose oxidase membrane was 0.34 units cm^?2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized d-glucose oxidase membrane was 1.6 × 10^?3 mol l^?1 and that of free enzyme was 4.8 × 10^?2 mol l^?1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized d-glucose oxidase membrane. The enzyme electrode responded linearly to d-glucose over the concentration 0–1000 mg dl^?1 within 10 s. When the enzyme electrode was applied to the determination of d-glucose in human serum, within day precision (CV) was 1.29% for d-glucose concentration with a mean value of 106.8 mg dl^?1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized d-glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of d-glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

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.     

Kinetics of ethanol production by immobilized cells of Zymomonas mobilis at varying d-glucose concentrations

Ethanol fermentation by cells of Zymomonas mobilis immobilized in calcium alginate gel has been studied using 5 to 30 wt% initial d-glucose in the medium. Up to 27% d-glucose was completely fermented and the maximum ethanol concentration of 12.6% (w/v) was obtained using an immobilized cell concentration of 58 g dry wt l^?1 of bead volume. The ethanol yield coefficient was almost unaffected by initial d-glucose concentration and its value was >95% of theoretical. The rates of ethanol production and d-glucose utilization first increased, with an increase in initial d-glucose concentration up to 13.6%, and then started to decrease upon a further increase in initial d-glucose concentration. Cell leakage from the calcium alginate beads was very low.     

Synthesis of phlorizin derivatives and their inhibitory effect on the renal sodium/d-glucose cotransport system

To characterize further the Na⁺/d-glucose cotransport system in renal brush border membranes, phlorizin - a potent inhibitor of d-glucose transport - has been chemically modified without affecting the d-glucose moiety or changing the side groups that are essential for the binding of phlorizin to the Na⁺/d-glucose cotransport system. One series of chemical modifications involved the preparation of 3-nitrophlorizin and the subsequent catalytic reduction of the nitro compound to 3-aminophlorizin. From 3-aminophlorizin, 3-bromoacetamido-, 3-dansyl- and 3-azidophlorizin have been synthesized. In another approach, 3′-mercuryphlorizin was obtained by reaction of phlorizin with Hg(II) acetate. The phlorizin derivatives inhibit sodium-dependent but not sodium-independent d-glucose uptake by hog renal brush border membrane vesicles in the following order of potency: 3′-mercuryphlorizin = phlorizin > 3-aminophlorizin > 3-bromoacetamidophlorizin > 3-azidophlorizin > 3-nitrophlorizin > 3-dansylphlorizin. 3-Bromoacetamidophlorizin - a potential affinity label - also inhibits sodium-dependent but not sodium-independent phlorizin binding to brush border membranes. In addition, sodium-dependent phosphate and sodium-dependent alanine uptake are not affected by 3-bromoacetamidophlorizin. The results described above indicate that specific modifications of the phlorizin molecule at the A-ring or B-ring are possible that yield phlorizin derivatives with a high affinity and high specificity for the renal Na⁺/d-glucose cotransport system. Such compounds should be useful in future studies using affinity labeling (3-bromoacetamido- and 3-azidophlorizin) or fluorescent probes (3-dansylphlorizin).     

Synthesis of phlorizin derivatives and their inhibitory effect on the renal sodium/d-glucose cotransport system

To characterize further the Na⁺/d-glucose cotransport system in renal brush border membranes, phlorizin - a potent inhibitor of d-glucose transport - has been chemically modified without affecting the d-glucose moiety or changing the side groups that are essential for the binding of phlorizin to the Na⁺/d-glucose cotransport system. One series of chemical modifications involved the preparation of 3-nitrophlorizin and the subsequent catalytic reduction of the nitro compound to 3-aminophlorizin. From 3-aminophlorizin, 3-bromoacetamido-, 3-dansyl- and 3-azidophlorizin have been synthesized. In another approach, 3′-mercuryphlorizin was obtained by reaction of phlorizin with Hg(II) acetate. The phlorizin derivatives inhibit sodium-dependent but not sodium-independent d-glucose uptake by hog renal brush border membrane vesicles in the following order of potency: 3′-mercuryphlorizin = phlorizin > 3-aminophlorizin > 3-bromoacetamidophlorizin > 3-azidophlorizin > 3-nitrophlorizin > 3-dansylphlorizin. 3-Bromoacetamidophlorizin - a potential affinity label - also inhibits sodium-dependent but not sodium-independent phlorizin binding to brush border membranes. In addition, sodium-dependent phosphate and sodium-dependent alanine uptake are not affected by 3-bromoacetamidophlorizin. The results described above indicate that specific modifications of the phlorizin molecule at the A-ring or B-ring are possible that yield phlorizin derivatives with a high affinity and high specificity for the renal Na⁺/d-glucose cotransport system. Such compounds should be useful in future studies using affinity labeling (3-bromoacetamido- and 3-azidophlorizin) or fluorescent probes (3-dansylphlorizin).     

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.     

Sensitivity of Na-coupled d-glucose uptake, Mg-ATPase and sucrase to perturbations of the fluidity of brush-border membrane vesicles induced by n-aliphatic alcohols

The aim of our work is to show the importance of the role of hydrophobic bonds in maintaining Mg²⁺-ATPase or sucrase activity and Na⁺-coupled d-glucose uptake normal for the brush border of rat enterocytes. The activity of the two enzymes and the d-glucose uptake were therefore measured under the action of n-aliphatic alcohols and related to the fluidity determined by ESR. Three concentrations were used for the first eight alcohols, those of octanol being about 1500-times lower than those of methanol. For each alcohol the d-glucose uptake and the fluidity were linear functions of the logarithm of the concentration, the linear regressions being practically parallel and equidistant. The concentrations (C) of the eight alcohols inhibiting the d-glucose uptake by 80% were similar to those increasing the membrane fluidity by 3%. The linear relationship which existed in both cases between log 1 / C and log P, P being octanol / water partition coefficients of the alcohols, was evidence of great sensitivity to the hydrophobic effect of the alcohols. Only the first alcohols, however, produced any notable inhibition of Mg²⁺-ATPase and sucrase. Hydrophobic bonds are thus shown to have little influence in maintaining the activity of Mg²⁺-ATPase and sucrase, but they modulate the Na⁺-coupled d-glucose uptake.     

Specificity of d-glucose transport by the apical membrane of Nereis diversicolor epidermis

(1) The specificity of d-[6-³H]glucose influx by a Na⁺-dependent and phlorizin-sensitive transport system in the apical epidermal membrane of the polychaete worm, Nereis diversicolor, was investigated in vivo. (2) The inhibitory effect of eleven d-glucose analogues on d-[6-³H]glucose influx from a 5 μM external concentration was recorded. The inhibitors (each tested at 5, 50, 500 and 5000 μM) were selected to illuminate the configurational requirements for interaction with the d-glucose transport system. (3) The following compounds were found to be significant inhibitors: methyl α-d-glucoside, methyl β-d-glucoside, d-galactose, $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$, 2-deoxy-d-glucose, d-xylose, myo-inositol, β-d-fructose; the effect was graded according to inhibitor concentration. l-Glucose also inhibited d-glucose influx but to the same extent at all four concentrations tested, suggesting transport site heterogeneity. d-Mannose and l-arabinose did not inhibit influx. (4) The most potent inhibitor, methyl-α-d-glucoside, was itself a substrate, and its transport was inhibited by phlorizin and d-glucose, as well as by substitution of Na⁺ in the incubation medium with Li⁺ or choline⁺. (5) We conclude that the specificity of the Na⁺-dependent d-glucose transporter in the apical epidermal membrane of Nereis is similar to that in the apical membrane of vertebrate small intestinal and proximal tubular epithelium, and in the tapeworm integument.     

Opposite effects of d-fructose on total versus cytosolic ATP/ADP ratio in pancreatic islet cells

d-fructose (10 mM) augments, in rat pancreatic islets, insulin release evoked by 10 mM d-glucose. Even in the absence of d-glucose, d-fructose (100 mM) displays a positive insulinotropic action. It was now examined whether the insulinotropic action of d-fructose could be attributed to an increase in the ATP content of islet cells. After 30-60 min incubation in the presence of d-glucose and/or d-fructose, the ATP and ADP content was measured by bioluminescence in either rat isolated pancreatic islets (total ATP and ADP) or the supernatant of dispersed rat pancreatic islet cells exposed for 30 s to digitonine (cytosolic ATP and ADP). d-fructose (10 and 100 mM) was found to cause a concentration-related decrease in the total ATP and ADP content and ATP/ADP ratio below the basal values found in islets deprived of exogenous nutrient. Moreover, in the presence of 10 mM d-glucose, which augmented both the total ATP content and ATP/ADP ratio above basal value, d-fructose (10 mM) also lowered these two parameters. The cytosolic ATP/ADP ratio, however, was increased in the presence of d-glucose and/or d-fructose. Under the present experimental conditions, a sigmoidal relationship was found between such a cytosolic ATP/ADP ratio and either ⁸⁶Rb net uptake by dispersed islet cells or insulin release from isolated islets. These data provide, to our knowledge, the first example of a dramatic dissociation between changes in total ATP content or ATP/ADP ratio and insulin release in pancreatic islets exposed to a nutrient secretagogue. Nevertheless, the cationic and insulinotropic actions of d-glucose and/or d-fructose were tightly related to the cytosolic ATP/ADP ratio.     

Sensitivity of Na+-coupled d-glucose uptake,Mg2+-ATPase and sucrase to perturbations of the fluidity of brush-border membrane vesicles induced by n-aliphatic alcohols

The aim of our work is to show the importance of the role of hydrophobic bonds in maintaining Mg²⁺-ATPase or sucrase activity and Na⁺-coupled d-glucose uptake normal for the brush border of rat enterocytes. The activity of the two enzymes and the d-glucose uptake were therefore measured under the action of $\text{n-}\text{aliphatic alcohols}$ and related to the fluidity determined by ESR. Three concentrations were used for the first eight alcohols, those of octanol being about 1500-times lower than those of methanol. For each alcohol the d-glucose uptake and the fluidity were linear functions of the logarithm of the concentration, the linear regressions being practically parallel and equidistant. The concentrations ($\text{C}$) of the eight alcohols inhibiting the d-glucose uptake by 80% were similar to those increasing the membrane fluidity by 3%. The linear relationship which existed in both cases between log 1 / $\text{C}$ and log $\text{P, P}$ being octanol / water partition coefficients of the alcohols, was evidence of great sensitivity to the hydrophobic effect of the alcohols. Only the first alcohols, however, produced any notable inhibition of Mg²⁺-ATPase and sucrase. Hydrophobic bonds are thus shown to have little influence in maintaining the activity of Mg²⁺-ATPase and sucrase, but they modulate the Na⁺-coupled d-glucose uptake.     

Examination of immobilized cells in a rotating packed drum reactor for the production of ethanol from d-glucose

A rotating packed drum reactor has been proposed as an immobilized whole cell reactor and its performance for ethanol production has been studied with yeast cells immobilized in calcium alginate gel. In a continuous operation with synthetic d-glucose medium containing 125 g d-glucose l^?1, ethanol productivity was 20 g l^?1 h^?1 at a space velocity of 0.38 l (l gel)^?1 h^?1. With intermittent aeration the viability of yeast cells after 270 h of operation remained above 65%. CO₂ removal was easy, but d-glucose conversion was low at a high space velocity.     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

The biguanide inhibition of d-glucose transport in membrane vesicles from small intestinal brush borders

Biguanides inhibit d-glucose uptake in vesicles from small-intestinal brush border membranes. Evidence is presented that this inhibition is due to a reduced concentration of Na⁺ in the microenvironment of the carrier(s) for d-glucose. Biguanides do not inhibit the uptake of either d-fructose or l-glucose.     

Time-course of altered islet phospholipids and of calcium binding and ionophoretic properties of islet lipids following glucose stimulation

The time-course of alteration in islet cell phospholipid content following d-glucose exposure in islet cells and in islet cell membranes was related to the ability of lipids extracted from both cultured pancreatic islet cells and from plasma membranes isolated from the islet cells to translocate calcium in two model membrane systems. The first model system (bulk-phase system) detected lipid species with the ability to bind calcium, irrespective of their ability to enhance calcium transport across cell membranes. The second system (multilamellar membrane system) detected lipid species with the ability to both bind calcium and to enhance calcium transport across cell membranes (true ionophores). Pre-exposure to high d-glucose concentration led to a rapid (within 1 min) fall in membrane phosphoinositides. This was partially blocked by mannoheptulose. A concurrent fall in calcium bindig activity of lipids from the plasma membrane was observed. In the whole islet cell fraction, d-glucose induced a marked increase in Ca²⁺ ionophoretic activity. Unlike the fall in membrane polyphosphoinositides and membrane Ca²⁺ binding activity, these changes were dependent on the presence of added extracellular calcium. l-Glucose was without effect on membrane phosphoinositide content. It is concluded that altered membrane and intracellular phospholipids may contribute to the increased availability of intracellular Ca²⁺ following d-glucose stimulation by virtue of theie Ca²⁺ binding and ionophoretic properties.     

Co-expression of d-glucose isomerase and d-psicose 3-epimerase: Development of an efficient one-step production of d-psicose

d-Psicose has been attracting attention in recent years because of its alimentary activities and is used as an ingredient in a range of foods and dietary supplements. To develop a one-step enzymatic process of d-psicose production, thermoactive d-glucose isomerase and the d-psicose 3-epimerase obtained from Bacillus sp. and Ruminococcus sp., respectively, were successfully co-expressed in Escherichia coli BL21 strain. The substrate of one-step enzymatic process was d-glucose. The co-expression system exhibited maximum activity at 65 °C and pH 7.0. Mg²⁺ could enhance the output of d-psicose by 2.32 fold to 1.6 g/L from 10 g/L of d-glucose. When using high-fructose corn syrup (HFCS) as substrate, 135 g/L d-psicose was produced under optimum conditions. The mass ratio of d-glucose, d-fructose, and d-psicose was almost 3.0:2.7:1.0, when the reaction reached equilibrium after an 8 h incubation time. This co-expression system approaching to produce d-psicose has potential application in food and beverage products, especially softdrinks.     

Chemical modification of the small intestinal Na+/d-glucose cotransporter by amino group reagents: Evidence for a role of amino group(s) in the binding of the sugar

Some amino group reagents inactivate the small-intestinal Na⁺/d-glucose cotransporter, as measured either as a catalyst of Na⁺-dependent d-glucose transport or as a Na⁺-dependent phlorizin ligand. The amino group(s) studied in this paper are not identical with those investigated previously (Biber, J., Weber, J. and Semenza, G. (1983) Biochim. Biochim. Acta 728, 429–437): these are protected from inactivation by the simultaneous presence of Na⁺ plus sugar substrates. They are thus likely to be located within the substrate-binding site.     

Triphenylmethylphosphonium uptake by pancreatic islet cells

Uptake of triphenylmethylphosphonium cation (TPMP⁺) was studied in pancreatic islet cells. Islets rich in β-cells were prepared from non-inbred ob/ob-mice and incubated with [³H]TPMP⁺ and l-[1-¹⁴C]glucose. Conjoined with the Nernst equation, the values for TPMP⁺ uptake in excess of the extracellular (l-glucose) space predicted membrane electric potentials far from those previously recorded with intracellular electrodes. Improved agreement with the electrode data was achieved by correcting for assumed voltage-independent binding of TPMP⁺; plausible correction terms were derived from the kinetics of TPMP⁺ efflux and from the uptake of [³H]TPMP⁺ in islets treated with non-radioactive TPMP⁺ at such a high concentration (50 μM) as to abolish the glucose oxidation. In whole islets the magnitude of the TPMP⁺-derived potentials decreased with increasing extracellular K⁺ in the range 5.9–130 mM, and was diminished by 20 mM d-glucose or 0.5 mM 2,4-dinitrophenol, but not by 20 mM 3-O-methyl-d-glucose, 20 mM d-mannoheptulose alone, or 10 μM chlorotetracycline. The effect of d-glucose was not observed in the presence of d-mannoheptulose and was diminished when 130 mM NaCl in the medium was replaced by sodium isethionate. The magnitude of TPMP⁺ uptake and the effects of K⁺ and dinitrophenol were reproduced with dispersed islet cells from ob/ob-mice and with whole islets of normal inbred NMRI-mice; the d-glucose effect was reproduced with NMRI-mouse islets. The results support our previous hypotheses that the depolarizing and insulin-releasing actions of d-glucose are in part mediated by electrodiffusion mechanisms involving K⁺ and Cl^?.     

Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process

A mathematical model for enzymatic cellulose hydrolysis, based on experimental kinetics of the process catalysed by a cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparation from Trichoderma longibrachiatum has been developed. The model takes into account the composition of the cellulase complex, the structural complexity of cellulose, the inhibition by reaction products, the inactivation of enzymes in the course of the enzymatic hydrolysis and describes the kinetics of d-glucose and cellobiose formation from cellulose. The rate of d-glucose formation decelerated through the hydrolysis due to a change in cellulose reactivity and inhibition by the reaction product, d-glucose. The rate of cellobiose formation decelerated due to inhibition by the product, cellobiose, and inactivation of enzymes adsorbed on the cellulose surface. Inactivation of the cellobiose-producing enzymes as a result of their adsorption was found to be reversible. The model satisfactorily predicts the kinetics of d-glucose and cellobiose accumulation in a batch reactor up to 70–80% substrate conversion on changing substrate concentration from 5 to 100 g l^?1and the concentration of the enzymic preparation from 5 to 60 g l^?1.     

Critical carboxyl group(s) in Na-dependent cotransporters of the intestinal brush-border membrane

We have previously provided functional evidence for a role of carboxyl group(s) in the mechanism of coupling of Na⁺ and d-glucose fluxes by the small-intestinal cotransporter(s) (Kessler, M. and Semenza, G. (1983) J. Membrane Biol. 76, 27–56). We present here a study on the inactivation of the Na⁺-dependent transport systems, but not of the Na⁺-independent ones, in the small-intestinal brush-border membrane, by hydrophobic carbodiimides. Although marginal or insignificant protection by the substrates or by Na⁺ was observed, the parallelism between Na⁺-dependence and inactivation by these carbodiimides strongly indicates the role of carboxyl group(s) previously indicated. Contrary to the carboxyl group identified by Turner ((1986) J. Biol. Chem. 261, 1041–1047) in the sugar binding site of the renal Na⁺/d-glucose cotransporter, the carboxyl group(s) studied here probably occur elsewhere in the cotransporter molecule.     

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

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