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At least 19 recordsLinked to original sources
PubMed1987

Re-examination of hexose-transporter inhibition and labelling by hexose isothiocyanates.

We have re-examined hexose-transport inhibition by hexose isothiocyanates and find that the inhibition is incomplete, probably because of decomposition of the reagent. The inhibition type is 'mixed', because hexose-transporter ligands such as maltose and cytochalasin B only give partial protection from inhibition. This suggests that a liganded-transporter-hexose isothiocyanate ternary complex is formed. We have compared the labelling of red-blood-cell membranes by [14C]MITC (D-maltose isothiocyanate) with the labelling obtained using a photoaffinity probe (ASA-BMPA [2-N-(4-azidosalicyloyl)-1,3-bis-(D-mannos-4'-yloxy)-2 -propylamine]) which gives specific labelling of the hexose transporter in band 4.5. [14C]MITC gives a partially D-glucose-displaceable labelling of a band 3 component in the same cell preparations which show ASA-BMPA labelling of band 4.5. This eliminates the possibility that band 4.5 labelling can only occur when the HITC (hexose isothiocyanate) binding protein in band 3 is proteolysed. HITC pretreatment does not decrease ASA-BMPA labelling of the exofacial site of band 4.5. This is also consistent with the formation of ternary complex. However, HITC pretreatment inhibits both reversible and photoactivated covalent [3H]cytochalasin B binding to band 4.5. These results suggest that, in the intact cell, interactions between a band 3 HITC-binding component and the inside cytochalasin B-binding site on the hexose transporter in band 4.5 may occur.

Affinity Labels↗
PubMed1990

Remodeling hexose-1-phosphate uridylyltransferase: mechanism-inspired mutation into a new enzyme, UDP-hexose synthase.

Hexose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-galactose and glucose-1-P with UDP-glucose and galactose-1-P by a double-displacement mechanism through a covalent intermediate (E-UMP), in which UMP is bonded to one of two histidine residues at the active site, H164 or H166. To identify which histidine is the nucleophilic catalyst, we prepared two specific mutants of the enzyme from Escherichia coli, H164G and H166G, in each of which the imidazole ring and methylene carbon of one histidine are deleted. To determine whether the function of the deleted imidazole in these mutants could be carried out by the imidazole ring in uridine 5'-(phosphoimidazolate) (UMP-Im), we examined the mutant proteins for catalytic activity in the reaction of UMP-Im with glucose-1-P to form UDP-glucose and imidazole. The mutant H166G catalyzes this reaction, as well as the reverse reaction, by a sequential kinetic mechanism involving ternary complexes as intermediates. The mutant enzyme also accepts galactose-1-P as a substrate to form UDP-galactose. Hexose-1-P uridylyltransferase does not catalyze these reactions, and H166G does not catalyze the wild-type reaction. The substrate Km values for the mutant enzyme are similar to those for hexose-1-P uridylyltransferase. The value of kcat in the direction of UDP-glucose formation is 1.31 +/- 0.01 s-1, compared with 350 s-1 for hexose-1-P uridylyltransferase, and in the reverse direction kcat is 4.8 +/- 0.4 s-1, compared with 960 s-1 for the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucosephosphates↗
PubMed1979

Stereospecific hexose transport by membrane vesicles from mouse fibroblasts: membrane vesicles retain increased hexose transport associated with viral transformation.

Membrane vesicles isolated from nontransformed BALB/c 3T3 mouse fibroblasts (3T3) and from those cells transformed by simian virus 40 (SV3T3) displayed carrier-mediated and stereospecific uptake of hexose as measured by the difference between D-[(14)C]glucose or its analogues and L-[(3)H]glucose uptake. Stereospecific uptake appeared to be linear for 5 sec and reached a maximum at 5-10 min. Stereospecific D-[(14)C]glucose uptake, osmotically sensitive and temperature dependent, was inhibited by unlabeled D-glucose or its analogues and was stimulated by the countertransport of accumulated unlabeled D-glucose. As with whole cells, the initial rate of stereospecific uptake by SV3T3 membrane vesicles was approximately 2.5-fold greater than that by 3T3 vesicles. Efflux of preloaded D-[(14)C]glucose was also faster from SV3T3 than from 3T3 membrane vesicles. The K(m) value was 5 mM for both the 3T3 and the SV3T3 membrane vesicles, but the V(max) values were 36 and 86 nmol/mg of protein per min, respectively, suggesting an increase in the number or availability of hexose carriers in transformed cell membranes. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicles. The binding of cytochalasin B to the SV3T3 membrane vesicles was significantly greater than that to 3T3 vesicles. Thus, the membrane vesicles retained many of the features of the altered hexose transport observed in whole cells in association with viral transformation.

Biological Transport, Active↗
PubMed1989

Rat brain hexokinase: further studies on the specificity of the hexose and hexose 6-phosphate binding sites.

Based on the lack of correlation between the ability of various hexoses to serve as substrate and the ability of the corresponding hexose 6-phosphates to inhibit brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), R. K. Crane and A. Sols (1954, J. Biol. Chem. 210, 597-606) proposed that this enzyme possesses two discrete sites capable of binding hexose moieties, one serving as the substrate binding site and a second, regulatory in function, to which inhibitory 6-phosphates bind. Subsequent work has provided further experimental support for this proposal. The pioneering work by Crane and Sols focused primarily on the specificity of these sites with respect to requirements for orientation of hydroxyl substituents at the various positions of the pyranose ring. The present study explores additional aspects of the specificity of these sites, namely, the effect of substitution of a sulfur atom in place of the oxygen in the pyranose ring on ability to serve as substrate or inhibitor, and the effect of modification in charge of the substituent at the 6-position on inhibitory effectiveness. 5-Thioglucose is a linear competitive (versus glucose) inhibitor of rat brain hexokinase, with a Ki of about 0.2 mM, and is a linear mixed inhibitor (versus ATP), with Ki values in this same range. 5-Thioglucose is not, however, readily phosphorylated by brain hexokinase. Thus, although 5-thioglucose binds with moderate affinity to the glucose binding site, it is not effectively used as a substrate of the enzyme. Inhibition of brain hexokinase by glucose 6-phosphate or its analogs has been found to require a dianionic substituent at the 6-position. The 6-fluorophosphate derivative and glucose 6-sulfate are poor inhibitors of the enzyme, and the Ki for inhibition by 1,5-anhydroglucitol 6-phosphate increases markedly at pH values below the pK of the 6-phosphate group, indicating that the monoanionic form is ineffective as an inhibitor. In contrast to the detrimental effect that substitution of the oxygen atom in the pyranose ring with a sulfur has on ability to serve as substrate, 5-thio analogs are considerably more effective as inhibitors, the Ki for inhibition by 5-thioglucose 6-phosphate being 10-fold lower than that seen with glucose 6-phosphate. This effect of the heteroatom substitution can partially offset the decreased inhibition resulting from monoanionic character at the 6-position, but the 6-fluorophosphate derivative of 5-thioglucose 6-phosphate still inhibits with a Ki about 1000-fold greater than that seen with 5-thioglucose 6-phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Hexose regulation of sodium-hexose transport in LLC-PK1 epithelia: the nature of the signal.

We have shown previously that the concentration of glucose in the growth medium regulates sodium-coupled hexose transport in epithelia formed by the porcine renal cell line LLC-PK1. Assayed in physiological salt solution, the ratio of the concentration of alpha-methyl glucoside (AMG) accumulated inside the cell at steady state to its concentration outside, and the number of glucose transporters, as measured by phlorizin binding, was inversely related to the glucose concentration in the growth medium. In this study, using a cloned line of LLC-PK1 cells, we provide evidence that the difference in AMG concentrating capacity is the result of a regulatory signal and not simply due to a selection process where the growth of cells with enhanced glucose transport is favored by low glucose medium or vice-versa. By adding glucose to conditioned medium (collected after 48 hr incubation with cells and therefore containing less than 0.1 mM glucose), we demonstrate that the signal in the growth medium is indeed the concentration of glucose rather than another factor secreted into or depleted from the medium. Fructose and mannose, two sugars not transported by the sodium-dependent glucose transporter, can substitute for glucose as a carbohydrate source in the growth medium and have a modest glucose-like effect on the transporter. Growth in medium containing AMG does not affect the transporter, indicating that the regulatory signal is not a direct effect of the hexose on its carrier but involves hexose metabolism.

Animals↗
PubMed1991

Hexose metabolism in pancreatic islets: enzyme-to-enzyme tunnelling of hexose 6-phosphates.

The fate of unlabelled D-glucose and D-[2-3H]glucose in pancreatic islets was simulated taking into account experimental values for glycolytic flux, intracellular concentration of D-glucose 6-phosphate and phosphoglucoisomerase activity. The model, which also takes into account the isotopic discrimination in velocity and intramolecular transfer of tritium between D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate in the reaction catalyzed by phosphoglucoisomerase, revealed that the predicted generation of 3HOH from D-[2-3H]glucose was much higher than the true experimental value. Such a discrepancy is reinforced by the consideration that the generation of 3HOH from D-[2-3H]glucose in islet cells is not solely attributable to the phosphoglucoisomerase-catalyzed detritiation of hexose 6-phosphates metabolized in the glycolytic pathway. In order to reconcile experimental and theoretical values for 3HOH production, it was found necessary to postulate enzyme-to-enzyme tunnelling of hexose 6-phosphates in the hexokinase/phosphoglucoisomerase/phosphofructokinase sequence. It is proposed that such a tunnelling may favour the anomeric specificity of D-glucose metabolism in islet cells, by restricting the anomerization of hexose 6-phosphates.

Animals↗
PubMed1988

Inhibition of hexose transport and labelling of the hexose carrier in human erythrocytes by an impermeant maleimide derivative of maltose.

Maltose-maleimide was synthesized as a potential affinity label for the facilitative hexose carrier with selectivity for exofacial sulphydryl groups. This reagent, although probably a mixture of isomers, did not significantly penetrate the plasma membrane of human erythrocytes at concentrations below 5 mM at 37 degrees C. When allowed to react to completion, it irreversibly inhibited the uptake of 3-O-methylglucose, with a half-maximal response at about 1.5-2.0 mM-reagent. The rate of transport inactivation was a saturable function of the maltose-maleimide concentration. Studies of reaction kinetics and effects of known transport inhibitors demonstrated that irreversible reaction occurred on the exofacial outward-facing carrier, although not at a site involved in substrate binding. Reaction of intact erythrocytes with [14C]maltose-maleimide resulted in labelling of a broad band 4.5 protein of Mr (average) 45,000-66,000 in electrophoretic gels. This protein was very likely the hexose carrier, since its labelling was inhibited by cytochalasin B. Exofacial band 4.5 labelling was stoichiometric with respect to transport inhibition, yielding an estimated 300,000 carriers/cell. These results suggest that the exofacial sulphydryl which reacts with maltose-maleimide is distinct from the substrate binding site on the hexose carrier, but that it confers substantial labelling selectivity to impermeant maleimides. Additionally, the high efficiency of carrier labelling obtained with maltose-maleimide is useful in quantifying numbers of carriers in whole cells.

Biological Transport↗
PubMed1981

Are lysosomes involved in hexose transport regulation? Turnover of hexose carriers and the activity of thiol cathepsins are arrested by cyanate and ammonia.

The cycloheximide-related loss of transport activity (manifested as a decrease in Vmax for transport) in cultured Nil hamster fibroblasts was blocked by the addition of carbamoyl phosphate, cyanate (a product of spontaneous phosphate elimination from carbamoyl phosphate), or ammonium salts to the culture medium. Acid proteases capable of hydrolyzing alpha-N-benzoyl-D,L-arginine-beta-naphthylamine (cathepsins B1, H, and L) were also inhibited in situ by ammonia and cyanate. The inactivation of these cathepsins by ammonia was irreversible and probably was related to the increase in the intralysosomal pH known to be caused by an accumulation of ammonia in the lysosomes. The inhibition of the cathepsin activity by cyanate in situ (and in cell-free extracts) was completely reversible and blocked irreversible inhibition of the cathepsin(s) by N-ethylmaleimide. The inactivation of the cathepsins caused by cyanate was deduced to be the result of reversible blocking of sulfhydryl groups essential to the thiol cathepsin activity. The concomitant inhibition of thiol cathepsins and hexose carrier inactivation provided further evidence for the involvement of lysosomal proteases in at least part of the mechanism that regulates the rate of hexose transport in animal cells.

Ammonia↗
PubMed1977

Synthesis of derivatives of 3-amino-2,3-dideoxy-L-hexoses related to daunosamine (3-amino-2,3,6-trideoxy-L-lyxo-hexose).

The synthesis is described of 3-amino-2,3-dideoxy-L-arabino-hexose (10), methyl 2,3-dideoxy-alpha-L-lyxo-hexopyranoside(17), methyl 3-amino-2,3-dideoxy-alpha-L-ribo-hexopyranoside (21), methyl 2,3-dideoxy-3-trifluoroacetamido-alpha-L-xylo-hexopyranoside (26), and certain derivatives from methyl 4,6-O-benzylidene-2-deoxy-alpha-L-arabino-hexopyranoside (3). Conversion of 2-deoxy-L-arabino-hexose into 3 by modified, standard procedures, and on a large scale, gave a 75% yield.

Daunorubicin↗
PubMed1988

Reaction of an exofacial sulfhydryl group on the erythrocyte hexose carrier with an impermeant maleimide. Relevance to the mechanism of hexose transport.

The presence of a reactive exofacial sulfhydryl on the human erythrocyte hexose carrier was used to test several predictions of the alternating conformation or one-site model of transport. The cell-impermeant glutathione-maleimide-I (GS-Mal) irreversibly inhibited hexose entry by decreasing the transport Vmax. This effect was potentiated by phloretin and maltose but decreased by cytochalasin B, indicating that under the one-site model the external sulfhydryl is on the outward-facing carrier but that it does not overlap with the exofacial substrate-binding site. Incubation of erythrocytes with maltose competitively inhibited the binding of [3H]cytochalasin B to the inward-facing carrier (Ki = 40 mM). Furthermore, both equilibrium cytochalasin B binding and its photolabeling of the band 4.5 carrier protein were decreased in ghosts prepared from GS-Mal-treated cells. Thus induction of an outward-facing carrier conformation with either maltose or GS-Mal caused the endofacial substrate-binding site to disappear. Dose-response studies of GS-Mal treatment of intact cells suggested that some functional carriers lack a reactive external sulfhydryl, which can be partially regenerated by pretreatment with excess cysteine. These data provide direct support for the one-site model of transport and further define the role of the external sulfhydryl in the transport mechanism.

3-O-Methylglucose↗
PubMed1990

Hexose metabolism in pancreatic islet cells: the coupling between hexose phosphorylation and mitochondrial respiration.

The possible relevance of D-glucose phosphorylation by mitochondria-bound hexokinase to the control of respiration was examined in mitochondria prepared from either tumoral pancreatic islet cells (RINm5F line) or normal rat liver. In both systems, ATP generated by mitochondria exposed to ADP and succinate could serve as a substrate for the phosphorylation of D-glucose. However, after exposure to exogenous ADP in the presence of succinate, only mitochondria isolated from RINm5F cells displayed a sizeable increase in O2 consumption in response to a subsequent administration of D-glucose. In this respect, the discrepancy between mitochondria from islet cells and liver, respectively, was found to be attributable to the much lower hexokinase activity, relative to respiratory rate, in liver than in RINm5F cell mitochondria. It is speculated that the coupling between hexose phosphorylation and respiration in islet cells may prime the mitochondria to generate ATP during the early metabolic and secretory response to a rise in extracellular D-glucose concentration.

Adenosine Diphosphate↗
PubMed1983

Preference of glucokinase for the alpha anomer of hexose: relation to alpha-anomer preference in hexose-induced insulin release by pancreatic islets.

The alpha anomers of glucose and mannose were more rapidly phosphorylated than the beta anomers by rat liver glucokinase, whose chromatographic and kinetic properties are known to be quite similar to those of pancreatic islet glucokinase. This result may explain our previous findings of the alpha-anomeric preference in glucose- and mannose-stimulated insulin release, and therefore suggests to support the hypothesis that glucokinase in islets functions as a crucial hexose sensing enzyme for insulin release induced by glucose and mannose.

Animals↗