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W F Widdas

Publications and source records attributed to W F Widdas.

At least 37 records · Page 2Linked to original sources
PubMed1988

Parameters for 3-O-methyl glucose transport in human erythrocytes and fit of asymmetric carrier kinetics.

1. Equilibrium exchanges in the range of 2-40 mM-3-O-methyl glucose at 16 degrees C suggested that the half-saturation concentration for exchange was 22 mM and that the maximum velocity (Vmax) was ca. 149 mmol l-1 min-1. 2. Initial rates of exchange influx from 1, 2, 4 and 8 mM into 76 mM solution gave a half-saturation value of 3.6 mM and a Vmax of 122 mmol-1 min-1. 3. The non-transportable inhibitor 4,6-O-ethylidene-alpha-D-glucopyranose (ethylidene glucose) acting on the outside of the cells inhibited 3-O-methyl glucose exchanges at 16 degrees C with an inhibition constant (KI) of ca. 11 mM. 4. Sen-Widdas exit experiments gave the half-saturation for 3-O-methyl glucose at 16 degrees C as only ca. 2 mM and the KI for ethylidene glucose as ca. 4 mM. 5. Efflux inhibitions by ethylidene glucose are satisfactorily predicted by the asymmetric carrier kinetics of Regen & Tarpley (1974) when using the parameters derived from the exchange experiments but not with parameters from Sen-Widdas exits. 6. Uphill transfer by counterflow experiments and Sen-Widdas exits cannot be fitted by the Regen and Tarpley kinetics (using the same parameters) unless the kinetics are modified to provide for an extra exchange element which replaces some of the net exit component in the equations. 7. At present the modification to the kinetics is only possible in computer simulations and data handling, but with it the fit to experimental results is good. The nature of the modification is described and in the light of it a revised interpretation of the significance of the Km derived from Sen-Widdas exits is discussed.

3-O-Methylglucose↗
PubMed1980

Asymmetry of hexose transfer system in erythrocytes of fetal and new-born guinea-pigs.

1. The asymmetries of affinities of two non-transportable competitive inhibitors of hexose transfer across fetal and new-born guinea-pig erythrocytes have been studied. 2. At 16 degrees C 4,6-O-ethylidene-alpha-D-glucopyranose (ethylidene glucose) inhibited 3-O-methyl glucose exchange at 20 mM with a K1 oc ca. 52 mM when present inside the cells and with a K1 oc ca. 10 mM when outside. This fivefold asymmetry is qualitatively similar to but smaller than the tenfold asymmetry of human erythrocytes (Baker, Basketter & Widdas, 1978). 3. Methyl-2,3-di-O-methyl-alpha-D-glucopyranoside (trimethyl glucoside) had K1 values of ca. 120 mM and ca. 160mM for inside and outside inhibition respectively. This is also qualitatively similar to the inhibition in human erythrocytes. 4. The inhibition produced by phlorizin, phloretin and Cytochalasin B was also studied in the erythrocytes of new-born guinea-pigs. The results were qualitatively similar to those for human erythrocytes but the inhibitory affinities were different. Thus while phlorizin and phloretin had higher affinities for the inhibition of exchange in new-born guinea-pig cells than human cells, the affinity of Cytochalasin B was less for new-born guinea-pig cells than for human cells. 5. It is concluded that the hexose transfer system in fetal and new-born guinea-pig red cells has asymmetric affinities similar to the system in human red cells but with different values of the inhibitory constants. The differences may represent species variations in a structural protein serving identical functions in the two species. 6. The possibility that fetal red cells with their facilitated transfer system play a role in sugar transport is discussed.

Animals↗
PubMed1978

Asymmetry of the hexose transfer system in human erythrocytes. Experiments with non-transportable inhibitors.

1. The asymmetrical nature of sugar affinity for the hexose transfer system in human red cells has been demonstrated using purified 4,6-O-ethylidene-alpha-D-glucopyranose (ethylidene glucose) to inhibit the exchange of glucose, 3-O-methyl glucose and galactose. 2. The half-saturation concentration for ethylidene glucose inside the cell is estimated at ca. 110 mM whereas on the outside the value for exchange inhibition is ca 11mM. 3. The asymmetrics of affinities of two related non-transportable inhibitors 1,2-O-isopropylidene-D-glucofuranose and methyl-2,3-di-O-methyl-alpha-D-glucopyranoside have also been studied. 4. From experiments at varying concentrations and on theoretical grounds the half-saturation concentration for non-transportable inhibitors on the outside surface is shown to be over-estimated by measuring inhibition of exchange. In consequence the actual asymmetry of affinities may be greater than observed. 5. Experiments with ethylidene glucose also suggest that conformational changes redistributing components of the hexose transfer system between inward and outward facing modes may occur.

Biological Transport↗
PubMed1978

Asymmetry of the hexose transfer system in human erythrocytes. Comparison of the effects of cytochalasin B, phloretin and maltose as competitive inhibitors.

1. Cytochalasin B inhibits glucose transfer in human red cells. With glucose exit the inhibition is typically non-competitive, but hexose exchange is competitively inhibited. 2. At 16 degrees C the inhibitory constant for inhibition of 3-O-methyl glucose exchange is estimated at 1.1 X 10(-7) M while that for inhibition of glucose exit is 5.0 X 10(-7) M. 3. Uptake of labelled Cytochalasin B includes a saturable component which when correlated with the inhibition of exchange corresponds to a maximal binding of ca. 2.4 X 10(5) molecules per cell. 4. The kinetic parameters are compared with those for maltose (a competitive inhibitor acting on the outside only) and phloretin (an inhibitor acting both inside and out). 5. Kinetic evidence suggests that Cytochalasin B reacts with the inside of the hexose transfer system and that the anomalous inhibitory characteristics are due to the chemical asymmetry of the system. Independent evidence in support of this view is discussed.

Binding, Competitive↗
PubMed1976

Sugars and sugar derivatives which inhibit the short-circuit current of the everted small intestine of the rat.

1. The short-circuit current of everted rat intestine supported on a perforated cannula proved to be stable for up to 3 hr and has been used to study competition between transportable and non-transportable sugars. 2. 4,6-O-Ethylidene-alpha-D-glucopyranose (ethylidene glucose) and 4,6-O-benzylidene-e alpha-D-glucopyranos (benzylinene glucose), two nontransportable inhibitors of the hexose transfer system in human erythrocytes, were found to reduce the short-circuit current generated by transportable sugars such as galactose or 3-O-methyl glucose. 3. These compounds were also found to reduce the basal short-circuit current established by the everted intestine in a sugar-free Krebs solution. Both types of inhibition approached saturation at the higher concentrations used. 4. Similar inhibitory properties were shown by mannose, a non-actively accumulated monosaccharide, and by the beta-disaccharides lactose and cellobiose. 5. It is suggested that this common pattern of behaviour is due to the ability of these compounds to react with the sites for active hexose transfer but without translocation by the system. The significance of the inhibition of the basal short-circuit current is briefly discussed in this context.

Animals↗
PubMed1973

The permeation of human red cells by 4,6-O-ethylidene- -D-glucopyranose (ethylidene glucose).

1. The glucose derivative 4, 6-O-ethylidene-alpha-D-glucopyranose (ethylidene glucose) was found to inhibit glucose exit competitively but its penetration into human red cells was unaffected by glucose in the medium.2. In penetrating red cells ethylidene glucose followed diffusion type kinetics without any evidence of saturation up to 360 mM. Besides its penetration being unaffected by glucose, 10(-5)M phloretin, which powerfully inhibits the facilitated transfer of hexoses, did not inhibit penetration.3. Red cells incubated with 1-fluoro-2, 4-dinitrobenzene (FDNB) until glucose exit was reduced by 95% showed no slowing of penetration by ethylidene glucose.4. The potentiation of the development of FDNB inhibition by sugars in the incubating medium was absent when ethylidene glucose was used and there was a slight protective action. Cells pre-incubated with 76 mM ethylidene glucose did not show an uphill transfer from 4 mM-[(14)C]glucose in the outside medium in contrast to cells pre-incubated in 76 mM glucose or in 76 mM 3-O-methyl glucose.5. The possibility that ethylidene glucose penetrated human red cells by simple diffusion was supported by its penetration of guinea-pig red cells at similar rates, by the occurrence of osmotic haemolysis in isosmotic solutions which was unaffected by copper ions and by the relatively high ether/water partition of the compound.

Animals↗
PubMed1973

The asymmetry of the facilitated transfer system for hexoses in human red cells and the simple kinetics of a two component model.

1. 4, 6-O-Ethylidene-alpha-D-glucopyranose (ethylidene glucose) has been used to study the competitive inhibition of glucose exchange fluxes when the reagent was (i) inside the cells and (ii) on the outside.2. 50% inhibition of glucose exchange at 20 mM and 16 degrees C required 200 mM ethylidene glucose when on the inside in contrast to 25-30 mM when on the outside.3. The inhibitions at different inhibitor/glucose concentration ratios were measured and analysis of the data suggested that the half-saturation constant for ethylidene glucose was 6 times that for glucose inside the cell as against 1.5 outside. The analysis, however, suggested an asymmetry in respect to the affinities for glucose of approximately ten-fold and this would make the asymmetry towards ethylidene glucose forty-fold.4. Such asymmetries make it necessary to consider a transfer mechanism for sugars with different components on the outer and inner membrane interfaces and simple kinetics for a two component system have been developed and used for analysing the experimental data quantitatively.5. The kinetic similarities to and difference from the kinetics of a simple mobile carrier and those of some more recent models are briefly discussed.

Biological Transport, Active↗
PubMed1968

Uptake of N-ethylmaleimide and I-Fluoro-2, 4-dinitrobenzene in relation to the irreversible inhibition of glucose transfer in the human erythrocyte.

1. The uptake of the inhibitors N-ethylmaleimide (NEM) and 1-fluoro-2,4-dinitrobenzene (DNFB) by human red cells has been correlated with the inhibition of glucose exit.2. With both inhibitors there was an initial rapid uptake by the cells with little inhibition; this was followed by a phase when inhibition was developing rapidly but uptake continued at a steady rate even after the development of inhibition had flattened off.3. The rate of uptake of DNFB during the rapid development of inhibition corresponded to about 4 x 10(8) molecules/cell for 100% inhibition, irrespective of the temperature of incubation. This cannot be used as an estimate of the number of glucose transfer sites in the cell membrane because of the lack of specificity.4. In an examination of lipids from red cells incubated with [(14)C]DNFB, labelling associated with lipids was eluted with peaks in chloroform-methanol 4:1 and 1:4 respectively. Thus, although DNFB is normally regarded as a protein reagent, involvement of lipids in the transfer of glucose could not be excluded.

Biological Transport, Active↗
PubMed1968

The effect of temperature on the competitive inhibition of glucose transfer in human erythrocytes by phenolphthalein, phloretin and stilboestrol.

1. Phenolphthalein, like other diphenols, has been shown to be a competitive inhibitor of the facilitated transfer system for glucose in the human erythrocyte.2. The concentration producing 50% inhibition is lower at low temperatures and increases steadily over the temperature range 10-40 degrees C. An Arrhenius plot of the results gives a slope of 19,300 cal/mole.3. The effect of temperature on inhibition by phloretin and stilboestrol has also been studied. The temperature variation of the concentration of phloretin giving 50% inhibition is similar to that for phenolphthalein, but that for stilboestrol is much less.4. There is a high cell/medium distribution ratio for stilboestrol, and the possibility that this may affect the temperature dependence of the aqueous concentrations required to give 50% inhibition is discussed.

Animals↗