Structural requirements for active intestinal transport. Spatial and bonding requirements at C-3 of the sugar.
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ABSTRACT:
SUBMITTER: Barnett JE
PROVIDER: S-EPMC1184929 | biostudies-other | 1969 Sep
REPOSITORIES: biostudies-other
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Project description:Several weakly transported sugars were tested for transport by the Na(+)-dependent sugar carrier with slices of everted hamster intestinal tissue. Sugars were assumed to be transported by this carrier if the accumulation was diminished in the absence of Na(+) and in the presence of the competitive inhibitor 1,5-anhydro-d-glucitol. The extent of accumulation was correlated with the number of hydroxyl groups in the d-gluco configuration if the ring oxygen was placed in the normal d-glucose position. 5-Thio-d-glucose, with a sulphur atom in the ring, was transported at about the same rate as d-glucose and had a similar K(i) for d-galactose transport, but myoinositol was poorly accumulated. It is suggested that there is no hydrogen bonding at the ring oxygen atom, but that the oxygen atom is found at this position as a result of steric constraints. No sugar without a hydroxyl group in the d-gluco position at C-2 of the sugar, including d-mannose, 2-deoxy-d-glucose, 2-chloro-2-deoxy-d-glucose and 2-deoxy-2-fluoro-d-glucose, was transported by the Na(+)-dependent carrier, but these sugars and l-fucose weakly and competitively inhibit the Na(+)-dependent accumulation of l-glucose into slices of everted hamster intestinal tissue. It is concluded that the bond between the carrier and C-2 of the sugar may be covalent, and a possible mechanism for active intestinal transport is proposed.
| S-EPMC1179295 | biostudies-other
Project description:1. A series of d-galactose derivatives substituted at C-1 and C-6 were tested for active accumulation by everted segments of hamster and rat intestine. 2. d-Galactose and 6-deoxy-6-fluoro-d-galactose were accumulated far more rapidly than 6-deoxy- and 6-chloro-6-deoxy-d-galactose, and this is interpreted as due to hydrogen-bonding at C-6 during the transport process. 3. 6-Bromo-6-deoxy- and 6-deoxy-6-iodo-d-galactose were not actively transported, indicating that the allowed size of substituent at C-6 lies between that of chlorine and bromine atoms. 4. Similar results were obtained at C-1. Both methyl alpha-d-galactopyranoside and methyl beta-d-galactopyranoside were well transported, but methyl beta-d-thiogalactopyranoside and 1-deoxy-d-galactose were not transported; d-galactopyranosyl fluoride was transported, but only poorly. Again hydrogen-bonding is suggested. 5. It is proposed that d-glucose is the ideal structure for active transport and that binding occurs at C-1, C-2, C-3, C-4 and C-6. Loss of two or more of these bonds usually causes loss of active transport. 6. By plotting Lineweaver-Burk plots of the rates of transport of the galactose derivatives, the apparent V and K(m) values were obtained. With hamster intestine both these values were very reproducible. Contrary to expectation, V varied for different sugars. 7. The K(i) of some of the analogues modified at C-1 and C-6 was determined with methyl alpha-d-glucoside as substrate. 8. An attempt to alkylate the carrier by using methyl 3,4-anhydro-alpha-d-galactoside was unsuccessful. There was no evidence that this compound was bound to the carrier.
| S-EPMC1186752 | biostudies-other
Project description:The structural requirements for binding to the glucose/sorbose-transport system in the human erythrocyte were explored by measuring the inhibition constants, K(i), for specifically substituted analogues of d-glucose when l-sorbose was the penetrating sugar. Derivatives in which a hydroxyl group in the d-gluco configuration was inverted, or replaced by a hydrogen atom, at C-1, C-2, C-3, C-4 or C-6 of the d-glucose molecule, all bound to the carrier, confirming that no single hydroxyl group is essential for binding to the carrier. The binding and transport of 1-deoxy-d-glucose confirmed that the sugars bind in the pyranose form. The relative inhibition constants of d-glucose and its deoxy, epimeric and fluorinated analogues are consistent with the combination of beta-d-glucopyranose with the carrier by hydrogen bonds at C-1, C-3, probably C-4, and possibly C-6 of the sugar. Both polar and non-polar substituents at C-6 enhance the affinity of d-glucose derivatives relative to d-xylose, and d-galactose derivatives relative to l-arabinose, and it is suggested that the carrier region around C-6 of the sugar may contain both hydrophobic and polar binding groups. The spatial requirements at C-1, C-2, C-3, C-4 and C-6 were explored by comparing the relative binding of d-glucose and its halogeno and O-alkyl substituents. The carrier protein closely approaches the sugar except at C-3 in the d-gluco configuration, C-4 and C-6. d-Glucal was a good inhibitor, showing that a strict chair form is not essential for binding. 3-O-(2',3'-Epoxypropyl)-d-glucose, a potential substrate-directed alkylating agent, bound to the carrier, but did not inactivate it.
| S-EPMC1177460 | biostudies-other
Project description:Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.
| S-EPMC4941443 | biostudies-literature
Project description:Nucleotide-sugar transporters (NSTs) are critical components of the cellular glycosylation machinery. They transport nucleotide-sugar conjugates into the Golgi lumen, where they are used for the glycosylation of proteins and lipids, and they then subsequently transport the nucleotide monophosphate byproduct back to the cytoplasm. Dysregulation of human NSTs causes several debilitating diseases, and NSTs are virulence factors for many pathogens. Here we present the first crystal structures of a mammalian NST, the mouse CMP-sialic acid transporter (mCST), in complex with its physiological substrates CMP and CMP-sialic acid. Detailed visualization of extensive protein-substrate interactions explains the mechanisms governing substrate selectivity. Further structural analysis of mCST's unique lumen-facing partially-occluded conformation, coupled with the characterization of substrate-induced quenching of mCST's intrinsic tryptophan fluorescence, reveals the concerted conformational transitions that occur during substrate transport. These results provide a framework for understanding the effects of disease-causing mutations and the mechanisms of this diverse family of transporters.
| S-EPMC6508934 | biostudies-literature
Project description:The Na(+)-dependent glucose transporter SGLT1 and the facilitated fructose transporter GLUT5 absorb sugars from the intestinal lumen across the brush-border membrane into the cells. The activity of these transport systems is known to be regulated primarily by diet and development. The cloning of these transporters has led to a surge of studies on cellular mechanisms regulating intestinal sugar transport. However, the small intestine can be a difficult organ to study, because its cells are continuously differentiating along the villus, and because the function of absorptive cells depends on both their state of maturity and their location along the villus axis. In this review, I describe the typical patterns of regulation of transport activity by dietary carbohydrate, Na(+) and fibre, how these patterns are influenced by circadian rhythms, and how they vary in different species and during development. I then describe the molecular mechanisms underlying these regulatory patterns. The expression of these transporters is tightly linked to the villus architecture; hence, I also review the regulatory processes occurring along the crypt-villus axis. Regulation of glucose transport by diet may involve increased transcription of SGLT1 mainly in crypt cells. As cells migrate to the villus, the mRNA is degraded, and transporter proteins are then inserted into the membrane, leading to increases in glucose transport about a day after an increase in carbohydrate levels. In the SGLT1 model, transport activity in villus cells cannot be modulated by diet. In contrast, GLUT5 regulation by the diet seems to involve de novo synthesis of GLUT5 mRNA synthesis and protein in cells lining the villus, leading to increases in fructose transport a few hours after consumption of diets containing fructose. In the GLUT5 model, transport activity can be reprogrammed in mature enterocytes lining the villus column. Innovative experimental approaches are needed to increase our understanding of sugar transport regulation in the small intestine. I close by suggesting specific areas of research that may yield important information about this interesting, but difficult, topic.
| S-EPMC1222226 | biostudies-other
Project description:Glycosylation is a fundamental cellular process that, in eukaryotes, occurs in the lumen of both the Golgi apparatus and the endoplasmic reticulum. Nucleotide sugar transporters (NSTs) are an essential component of the glycosylation pathway, providing the diverse range of substrates required for the glycosyltransferases. NSTs are linked to several developmental and immune disorders in humans, and in pathogenic microbes they have an important role in virulence. How NSTs recognize and transport activated monosaccharides, however, is currently unclear. Here we present the crystal structure of an NST, the GDP-mannose transporter Vrg4, in both the substrate-free and the bound states. A hitherto unobserved requirement of short-chain lipids in activating the transporter supports a model for regulation within the highly dynamic membranes of the Golgi apparatus. Our results provide a structural basis for understanding nucleotide sugar recognition, and provide insights into the transport and regulatory mechanism of this family of intracellular transporters.
| S-EPMC5701743 | biostudies-literature
Project description:1. In the yeast Rhodotorula gracilis several amino sugars were actively transported. Glucosamine, which is largely protonated at physiological pH (pK 7.75) was used as a model substrate. At pH 6.75 its half-saturation constant was 1 mM and the maximal velocity was 50 nmol/min per mg dry wt. 2. Amino sugars were taken up via the monosaccharide carrier. The transport of glucosamine was strongly restricted by monosaccharides. D-Xylose inhibited competitively the uptake of glucosamine. The inhibition constant was 1 mM. Cells preloaded with D-xylose showed exchange transport on subsequent addition of glucosamine. 3. Transport of glucosamine was energized by the membrane potential. Uncoupling agents such as carbonyl cyanide m-chlorophenyl-hydrazone and the lipophilic cation TPP+ (tetraphenylphosphonium ion) at concentrations that depolarized the membrane potential inhibited the uptake of glucosamine. Conversely the transport of glucosamine partly dissipated the membrane potential, which was monitored by radioactively labelled lipophilic cations. 4. The translocated charges were electrically compensated by the extrusion of protons and K+ (1 glucosamine molecule/0.85 H+ + 0.15 K+). 5. An increase of the pH in the range 4.75-8.75 lead to a decrease of the half-saturation constant from 5 mM to 1 mM and to an optimum of the maximal velocity at pH 6.75. We suggest that this fair constancy is due to the carrier not distinguishing between the protonated form of glucosamine (pH less than 7.75) and the deprotonated form (pH greater than 7.75). The increase of V(T) (maximal transport velocity) between pH 4.75 and 6.75 is due to the increase of the membrane potential: the decrease between pH 6.75 and 8.75 is due to the deprotonization of the carrier.
| S-EPMC1162766 | biostudies-other
Project description:Intestinal vitamin C (Asc) absorption was believed to be mediated by the Na(+)-dependent ascorbic acid transporter SVCT1. However, Asc transport across the intestines of SVCT1 knock-out mice is normal indicating that alternative ascorbic acid transport mechanisms exist. To investigate these mechanisms, rodents were gavaged with Asc or its oxidized form dehydroascorbic acid (DHA), and plasma Asc concentrations were measured. Asc concentrations doubled following DHA but not Asc gavage. We hypothesized that the transporters responsible were facilitated glucose transporters (GLUTs). Using Xenopus oocyte expression, we investigated whether facilitative glucose transporters GLUT2 and GLUT5-12 transported DHA. Only GLUT2 and GLUT8, known to be expressed in intestines, transported DHA with apparent transport affinities (Km) of 2.33 and 3.23 mm and maximal transport rates (Vmax) of 25.9 and 10.1 pmol/min/oocyte, respectively. Maximal rates for DHA transport mediated by GLUT2 and GLUT8 in oocytes were lower than maximal rates for 2-deoxy-d-glucose (Vmax of 224 and 32 pmol/min/oocyte for GLUT2 and GLUT8, respectively) and fructose (Vmax of 406 and 116 pmol/min/oocyte for GLUT2 and GLUT8, respectively). These findings may be explained by differences in the exofacial binding of substrates, as shown by inhibition studies with ethylidine glucose. DHA transport activity in GLUT2- and GLUT8-expressing oocytes was inhibited by glucose, fructose, and by the flavonoids phloretin and quercetin. These studies indicate intestinal DHA transport may be mediated by the facilitative sugar transporters GLUT2 and GLUT8. Furthermore, dietary sugars and flavonoids in fruits and vegetables may modulate Asc bioavailability via inhibition of small intestinal GLUT2 and GLUT8.
| S-EPMC3610982 | biostudies-literature
Project description:IntroductionModern, urban lifestyles have engineered physical activity out of everyday life and this presents a major threat to human health. The Netherlands is a world leader in active travel, particularly cycling, but little research has sought to quantify the cumulative amount of physical activity through everyday walking and cycling.MethodsUsing data collected as part of the Dutch National Travel Survey (2010 - 2012), this paper determines the degree to which Dutch walking and cycling contributes to meeting minimum level of physical activity of 150 minutes of moderate intensity aerobic activity throughout the week. The sample includes 74,465 individuals who recorded at least some travel on the day surveyed. As physical activity benefits are cumulative, all walking and cycling trips are analysed, including those to and from public transport. These trips are then converted into an established measure of physical activity intensity, known as metabolic equivalents of tasks. Multivariate Tobit regression models were performed on a range of socio-demographic, transport resources, urban form and meteorological characteristics.ResultsThe results reveal that Dutch men and women participate in 24 and 28 minutes of daily physical activity through walking and cycling, which is 41% and 55% more than the minimum recommended level. It should be noted however that some 57% of the entire sample failed to record any walking or cycling, and an investigation of this particular group serves as an important topic of future research. Active transport was positively related with age, income, bicycle ownership, urban density and air temperature. Car ownership had a strong negative relationship with physically active travel.ConclusionThe results of this analysis demonstrate the significance of active transport to counter the emerging issue of sedentary lifestyle disease. The Dutch experience provides other countries with a highly relevant case study in the creation of environments and cultures that support healthy, active living.
| S-EPMC4388541 | biostudies-literature