Phlorizin recognition in a C-terminal fragment of SGLT1 studied by tryptophan scanning and affinity labeling.
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SGLT1 as a sodium/glucose cotransporter is strongly inhibited by phlorizin, a phloretin 2'-glucoside that has strong interactions with the C-terminal loop 13. We have examined phlorizin recognition by the protein by site-directed single Trp scanning mutagenesis experiments. Six mutants (Q581W, E591W, R601W, D611W, E621W, and L630W) of truncated loop 13 (amino acids 564-638) were expressed in Escherichia coli and purified to homogeneity. Changes in Trp quenching and positions of the emission maxima were determined after addition of phlorizin. D611W displayed the largest quenching of 80%, followed by R601W (67%). It also exhibited the maximum red shift in Trp fluorescence ( approximately 14 nm), indicating an exposure of this region to a more hydrophilic environment. Titration experiments performed for each mutant showed a similar affinity for all mutants, except for D611W, which exhibited a significantly lower affinity (Kd approximately 54 microm). Also the maximum change in the collisional quenching constant by acrylamide was noted for D611W (KSV = 11 m-1 in the absence of phlorizin and 55 m-1 in its presence). Similar results were obtained with phloretin. CD measurements and computer modeling revealed that D611W is positioned in a random coil situated between two alpha-helical segments. By combining gel electrophoresis, enzymatic fragmentation, and matrix-assisted laser desorption ionization mass spectrometry, we also analyzed truncated loop 13 photolabeled with 3-azidophlorizin. The attachment site of the ortho-position of aromatic ring B of phlorizin was localized to Arg-602. Taken together, these data indicate that phlorizin binding elicits changes in conformation leading to a less ordered state of loop 13. Modeling suggests an interaction of the 4- and 6-OH groups of aromatic ring A of phlorizin with the region between amino acids 606 and 611 and an interaction of ring B at or around amino acid 602. Phloretin seems to interact with the same region of the protein. (+info)
Substrate specificity of small-intestinal lactase. Assessment of the role of the substrate hydroxyl groups.
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Lactase-phlorizin hydrolase is a disaccharidase present in the small intestine of mammals. This enzyme has two active sites, one being responsible for the hydrolysis of lactose. Lactase activity is thought to be selective towards glycosides with a hydrophilic aglycon. In this work, we report a systematic study on the importance of each hydroxyl group in the substrate molecule for lactase activity. For this purpose, all of the monodeoxy derivatives of methyl beta-lactoside and other lactose analogues are studied as lactase substrates. With respect to the galactose moiety, it is shown here that HO-3' and HO-2' are necessary for hydrolysis of the substrates by lactase. Using these chemically modified substrates, it has been confirmed that lactase does not behave as a typical beta-galactosidase, since it does not show an absolute selectivity with respect to substitution and stereochemistry at C4' in the galactose moiety of the substrate. However, the glucose moiety, in particular the HO-6, appears to be important for substrate hydrolysis, although none of the hydroxyl groups seemed to be essential. In order to differentiate both activities of the enzyme, a new assay for the phlorizin-hydrolase activity has also been developed. (+info)
ION TRANSPORT IN ISOLATED RABBIT ILEUM. II. THE INTERACTION BETWEEN ACTIVE SODIUM AND ACTIVE SUGAR TRANSPORT.
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The addition of actively transported sugars to the solution bathing the mucosal surface of an in vitro preparation of distal rabbit ileum results in a rapid increase in the transmural potential difference, the short-circuit current, and the rate of active Na transport from mucosa to serosa. These effects are dependent upon the active transport of the sugar per se and are independent of the metabolic fate of the transported sugar. Furthermore, they are inhibited both by low concentrations of phlorizin in the mucosal solution and by low concentrations of ouabain in the serosal solution. The increase in the short-circuit current, DeltaI(sc), requires the presence of Na in the perfusion medium and its magnitude is a linear function of the Na concentration. On the other hand, DeltaI(sc) is a saturable function of the mucosal sugar concentration which is consistent with Michaelis-Menten kinetics suggesting that the increase in active Na transport is stoichiometrically related to the rate of active sugar transport. An interpretation of these findings in terms of a hypothetical model for intestinal Na and sugar transport is presented. (+info)
THE EFFECTS OF ADRENALINE AND OTHER DRUGS AFFECTING CARBOHYDRATE METABOLISM ON CONTRACTIONS OF THE RAT DIAPHRAGM.
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The ability of several drugs to restore directly elicited twitches of the rat diaphragm depressed by excess potassium chloride has been studied. The drugs found to be effective were sympathomimetic amines, insulin, glucagon, caffeine, theophylline, calcium chloride and hexosephosphates. The effects of the sympathomimetic amines and glucagon were blocked by beta-receptor blocking agents. Phloridzin blocked the effect of insulin and depressed that of glucagon. The increase in twitch tension still occurred under anaerobic conditions and was not abolished by the glycolytic inhibitor, iodoacetate. All of the effective drugs are known to affect carbohydrate metabolism and the suggestion by Ellis (1955) that the effect on contractions may be a result of increased intracellular hexosephosphate levels is discussed. (+info)
ULTRASTRUCTURE OF ISOLATED KIDNEY MITOCHONDRIA TREATED WITH PHLORIZIN AND ATP.
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Direct electron microscopic evidence is reported of the ultrastructure of mitochondrial membranes and compartments in mitochondria isolated in 0.5 M sucrose from the rat kidney cortex and the experimental changes they undergo with phlorizin and ATP treatment. A heterogeneous population of mitochondria is recognized under control conditions. The mitochondria appear to be of 3 main types, normal, swollen, and contracted. Under phlorizin treatment, most of the mitochondria swell in less than 15 minutes, apparently at the expense of the matrix. Treatment with ATP, on the other hand, produces, during the same time, a marked contraction of the isolated mitochondria, with many refoldings of the inner membrane and marked increase in the electron opacity of the matrix. It is concluded from these observations that mitochondrial swelling and contraction should be related mainly to the matrix content. (+info)
Localization of the phlorizin site on Na, K-ATPase in red cell membranes.
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Phlorizin at 2 X 10(-4) M inhibited Na+ and Rb+-activated ATPase activities in human red cell membranes by 43%. It inhibited the 86Rb uptake activity of erythrocytes by only 15%. 86Rb uptake into resealed ghosts was inhibited strongly when phlorizin and ATP were preloaded in the ghosts before resealing. Na,K-ATPase activity in the resealed ghosts was also inhibited in the presence of phlorizin inside but not outside the ghosts. These findings suggested that the phlorizin site is located inside the cell. (+info)
Inhibition of ascorbic acid transport in human neutrophils by glucose.
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Because of the structural similarity between glucose and ascorbic acid, we investigated the effect of glucose on uptake and accumulation of ascorbic acid in isolated normal human neutrophils. Ascorbic acid accumulation was determined using high-performance liquid chromatography with coulometric electrochemical detection, in conjunction with liquid scintillation spectrometry. Ascorbic acid accumulation in neutrophils is mediated by a high and a low affinity transport activity. In neutrophils from different volunteers, glucose inhibited uptake and accumulation of ascorbic acid by both transport activities 3-9-fold. The mechanism of inhibition was different for each transport activity: inhibition of the high affinity transport activity was noncompetitive, while inhibition of the low affinity activity was competitive. Glucose-induced inhibition of both ascorbic acid transport activities occurred in neutrophils of all donors tested and was fully reversible. Although the mechanism of ascorbic acid accumulation appeared to be different than that for glucose transport, other monosaccharides and glucose transport inhibitors also inhibited ascorbic acid accumulation. These are the first data to suggest that ascorbic acid accumulation in neutrophils can be regulated by compounds of similar structure. (+info)
Sugar uptake into brush border vesicles from normal human kidney.
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Uptake studies of simple sugars were performed on a membrane fractions containing osmotically active vesicles prepared from normal human kidney cortex. The uptake of D-glucose was found to be sodium-dependent and phlorizin-sensitive. The specificity of the D-glucose transport mechanism is such that it is shared by alpha-methyl-D-glucoside, D-galactose, and 5-thio-D-glucose, while 2-deoxy-D-glucose, 3-O-methyl-D-glucose, D-mannose, and D-fructose show little, if any, affinity. Measurement of the sodium-dependent component of the initial D-glucose uptake as a function of glucose concentration resulted in a curvilinear Scatchard plot, indicating the possibility of cooperative effects, or alternatively, the existence of two (or more) sodium-dependent D-glucose transporters. In the case of two transporters, we estimate that Km congruent to 0.3 mM and Vmax congruent to 2.5 nmol/min per mg of protein for the "high-affinity transporter," and Km approximately 6 mM and Vmax approximately 8 nmol/min per mg of protein for the "low-affinity transporter." These specificity and kinetic properties strongly suggest that the sodium-dependent D-glucose transport mechanism characterized in our studies is localized to the brush border of the proximal tubule. (+info)