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PubMed1982

Aspects of maltose transport in Escherichia coli: established facts and educated guesses.

The transport system can translocate maltose and maltodextrins (up to 7 glucose moieties) in chemically unmodified form against concentration gradients that can reach 1:10(5). To overcome the problems of diffusion of substrate across the outer membrane at substrate concentrations below 0.1 mM, the receptor for phage lambda has to be present in the outer membrane. The facilitated diffusion via the lambda receptor is accomplished by its interaction with maltose-binding protein. The maltose-binding protein-substrate complex, but not free substrates is recognized at the cytoplasmic membrane. This implies that the maltose-binding protein is essential for substrate translocation and that the cytoplasmic membrane has no second maltodextrin recognition site. Substrate translocation through the cytoplasmic membrane is tightly coupled to energy consumption and unidirectionally inward. The energy source used for substrate accumulation is not the electrochemical potential of protons, but phosphate-bound energy, likely to be ATP. In mutants that are blocked in maltose metabolism, exit of maltose occurs in chemically modified form as acetylmaltose. It is energy-dependent, blocked by uncouplers of the proton-conductive type and is stimulated by energy sources. It is not mediated by any malB-dependent function. The membrane-bound components of the system may establish a non-specific pore that is triggered by its interaction with the maltose-binding protein bound to its substrate. After translocation of one molecule substrate the pore has to be re-energized, possibly by ATP hydrolysis.

ATP-Binding Cassette Transporters↗
PubMed1960

Tracer exchange vs. net uptake of glucose through human red cell surface. New evidence for carrier-mediated diffusion.

Previous kinetic studies of net sugar movements through the human erythrocyte surface (in response to concentration gradients) have led to postulation of a special "carrier" system for transfer of monosaccharides in these cells. But alternatively some sort of non-specific depression of cell permeability at high sugar concentrations has been suggested as a possible basis for the saturation kinetics and the competitive phenomena observed. New theoretical calculations show that these two interpretations predict entirely different orders of magnitude for the relative rate of tracer glucose exchange at such high sugar levels. Therefore, the speeds of gross chemical equilibration and of tracer glucose equilibration were compared by means of serial analyses on quickly separated cells and media, in thick red cell suspensions. Glucose was first added to glucose-free suspensions, and its entry into the cells followed; then C14-glucose was added after attainment of chemical equilibrium, and the tracer equilibration similarly followed. The speed of the tracer movement in relation to the speed of net uptake was on the order of 50 to 100 times greater than would be found in an uncomplicated diffusion process, regardless of what depressant effect might be occasioned by the high sugar levels. In contrast, the comparative rates observed are predicted by the previously proposed facilitated-diffusion mobile-carrier model for monosaccharide transfer, if the glucose-carrier complex is assigned a dissociation constant (at 20 degrees C.) in the neighborhood of 1 mM.

Biological Transport↗
PubMed1987

Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon.

The recent demonstration that the human colon adenocarcinoma cell line Caco-2 was susceptible to spontaneous enterocytic differentiation led us to consider the question as to whether Caco-2 cells would exhibit sodium-coupled transport of sugars. This problem was investigated using isotopic tracer flux measurements of the nonmetabolizable sugar analog alpha-methylglucoside (AMG). AMG accumulation in confluent monolayers was inhibited to the same extent by sodium replacement, 200 microM phlorizin, 1 mM phloretin, and 25 mM D-glucose, but was not inhibited further in the presence of both phlorizin and phloretin. Kinetic studies were compatible with the presence of both a simple diffusive process and a single, Na+-dependent, phlorizin- and phloretin-sensitive AMG transport system. These results also ruled out any interaction between AMG and a Na+-independent, phloretin-sensitive, facilitated diffusion pathway. The brush-border membrane localization of the Na+-dependent system was inferred from the observations that its functional differentiation was synchronous with the development of brush-border membrane enzyme activities and that phlorizin and phloretin addition 1 hr after initiating sugar transport produced immediate inhibition of AMG uptake as compared to ouabain. Finally, it was shown that brush-border membrane vesicles isolated from the human fetal colonic mucosa do possess a Na+-dependent transport pathway(s) for D-glucose which was inhibited by AMG and both phlorizin and phloretin. Caco-2 cells thus appear as a valuable cell culture model to study the mechanisms involved in the differentiation and regulation of intestinal transport functions.

Adenocarcinoma↗
PubMed2006

Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H+ symporter.

Candida intermedia PYCC 4715 was previously shown to grow well on xylose and to transport this sugar by two different transport systems: high-capacity and low-affinity facilitated diffusion and a high-affinity xylose-proton symporter, both of which accept glucose as a substrate. Here we report the isolation of genes encoding both transporters, designated GXF1 (glucose/xylose facilitator 1) and GXS1 (glucose/xylose symporter 1) respectively. Although GXF1 was isolated by functional complementation of an HXT-null (where Hxt refers to hexose transporters) Saccharomyces cerevisiae strain, isolation of the GXS1 cDNA required partial purification and micro-sequencing of the transporter, identified by its relative abundance in cells grown on low xylose concentrations. Both genes were expressed in S. cerevisiae and the kinetic parameters of glucose and xylose transport were determined. Gxs1 is the first yeast xylose/glucose-H+ symporter to be characterized at the molecular level. Comparison of its amino acid sequence with available sequence data revealed the existence of a family of putative monosaccharide-H+ symporters encompassing proteins from several yeasts and filamentous fungi.

Amino Acid Sequence↗
PubMed1991

D-glucose permeability in river lamprey (Lampetra fluviatilis) and carp (Cyprinus carpio) erythrocytes.

1. The transport of 3-O-methyl-D-glucose (3-OMG) in lamprey and carp erythrocytes was studied. 2. In lamprey erythrocytes the half-time for the equilibration of 3-OMG was fast, approx. 8 min. In contrast, the erythrocytes of carp were almost impermeable to 3-OMG, with a half-time for equilibration of 14.2 hr. 3. 3-OMG was taken up by lamprey erythrocytes both by facilitated diffusion and simple diffusion. The presence of carrier-mediated transport was indicated by saturation kinetics and by inhibition by phloretin. The Km and Vm of the saturable component of 3-OMG transport were 1.6 mmol/l and 12.4 mmol/kg packed cells/hr. 4. Since the 3-OMG transport in carp erythrocytes showed no sign of saturation kinetics, it appears to proceed by simple diffusion only. 5. These results suggest that, as for most other teleost fish so far studied, low glucose permeability is insufficient to maintain normal energy metabolism in carp erythrocytes. In contrast, in agnathans facilitated glucose transport seems to be quite effective.

3-O-Methylglucose↗
PubMed2004

How do site-specific DNA-binding proteins find their targets?

Essentially all the biological functions of DNA depend on site-specific DNA-binding proteins finding their targets, and therefore 'searching' through megabases of non-target DNA. In this article, we review current understanding of how this sequence searching is done. We review how simple diffusion through solution may be unable to account for the rapid rates of association observed in experiments on some model systems, primarily the Lac repressor. We then present a simplified version of the 'facilitated diffusion' model of Berg, Winter and von Hippel, showing how non-specific DNA-protein interactions may account for accelerated targeting, by permitting the protein to sample many binding sites per DNA encounter. We discuss the 1-dimensional 'sliding' motion of protein along non-specific DNA, often proposed to be the mechanism of this multiple site sampling, and we discuss the role of short-range diffusive 'hopping' motions. We then derive the optimal range of sliding for a few physical situations, including simple models of chromosomes in vivo, showing that a sliding range of approximately 100 bp before dissociation optimizes targeting in vivo. Going beyond first-order binding kinetics, we discuss how processivity, the interaction of a protein with two or more targets on the same DNA, can reveal the extent of sliding and we review recent experiments studying processivity using the restriction enzyme EcoRV. Finally, we discuss how single molecule techniques might be used to study the dynamics of DNA site-specific targeting of proteins.

DNA↗
PubMed1980

Sugar transport by larval and adult Proterometra macrostoma (Digenea) in relation to environmental factors.

Monosaccharide transport systems were identified in rediae and cercariae of Proterometra macrostoma. Glucose transport by cercariae in vitro was accomplished by the bodies, but tails absorbed glucose by diffusion alone. No sugar transport system was detected in adults obtained from laboratory infections of sunfish. Temperature of Elkhorn Creek where infected snails (Goniobasis livescens) were found varies from 28 C in summer to 5 C in winter. Glucose transport by both larval stages was optimal between 30 and 25 C and was negligible below 15 C. Transport by rediae was activated by Na+ and was maximal at 50 mM, the approximate Na+ concentration of snail hemolymph. The redial glucose transport system was inhibited by various sugars and was more sensitive to phlorizin than to phloretin. Rediae accumulated glucose and nonmetabolized 3-O-methyl-glucose against apparent concentration gradients, indicating an active transport system. In contrast, glucose transport by "embryonic" cercariae was completely inhibited by 10 mM Na+. Transport by cercariae aged in creek, water 6 hr became relatively insensitive to Na+, and were less permeable to glucose by diffusion. The cercarial system differed in other characteristics from the redial system, including its high sensitivity to phloretin relative to phlorizin and its lower Vmax. The cercarial system apparently functioned only as a facilitated diffusion system that served to move sugar across the tegument down its chemical gradient. Development of P. macrostoma thus showed functional transformations of the tegument that may be subject to regulation by environmental factors.

Animals↗
PubMed1987

Lipid fluidity and membrane protein dynamics.

Membrane fluidity plays an important role in cellular functions. Membrane proteins are mobile in the lipid fluid environment; lateral diffusion of membrane proteins is slower than expected by theory, due to both the effect of protein crowding in the membrane and to constraints from the aqueous matrix. A major aspect of diffusion is in macromolecular associations: reduction of dimensionality for membrane diffusion facilitates collisional encounters, as those concerned with receptor-mediated signal transduction and with electron transfer chains. In mitochondrial electron transfer, diffusional control is prevented by the excess of collisional encounters between fast-diffusing ubiquinone and the respiratory complexes. Another aspect of dynamics of membrane proteins is their conformational flexibility. Lipids may induce the optimal conformation for catalytic activity. Breaks in Arrhenius plots of membrane-bound enzymes may be related to lipid fluidity: the break could occur when a limiting viscosity is reached for catalytic activity. Viscosity can affect protein conformational changes by inhibiting thermal fluctuations to the inner core of the protein molecule.

Diffusion↗
PubMed1975

Orthophosphate transport in the erythrocyte of normal subjects and of patients with X-linked hypophosphatemia.

We have examined the mechanism of TCA-soluble orthophosphate (Pi) transfer across the membrane of mature human erythrocytes in normal subjects and in patients with X-linked hypophosphatemia (X-LH). The studies were carried out largely at pH 7.4 and 37 degrees C, in partial stimulation of conditions in vivo. (a) At physiological concentrations (1-2 mM) Pi enters the intact normal erythrocyte down its chemical gradient and under no conditions could we identify a steady-state trans-membrane gradient for Pi greater than 0.6. Calculations of the phosphate anion distribution ratio using the Nernst equation yield theoretical values that closely approximate observed values. (b) Glycolytic inhibitors have little effect on total entry of 32Pi inti erythrocytes but they do affect the intracellular distribution of Pi. In the presence of iodoacetamide, label accumulates almost exclusively in the orthophosphate pool and less than 1% enters the organic phosphate pool. (c) Specific activity measurements in unblocked cells indicate that Pi anion equilibrates first with its intracellular Pi pool. These initial findings imply that neither group translocation, nor energy coupling, influence Pi permeation into the human erythrocytes. (d) The relationship between 32P entry and extracellular Pi concentration is parabolic in the presence of chloride, and linear in the presence of sulfate. The kinetics of concentration dependent entrance cannot be examined and saturability of Pi entry cannot be identified under these conditions. (e) The competitive inhibitor arsenate partially inhibits the initial rate and steady-state flux of orthophosphate in erythrocytes treated with iodoacetamide to inhibit glycolysis. However, a significant portion of Pi transport escapes arsenate inhibition. (f) Activation energies for Pi entry, in nonglycolizing erythrocytes are much higher than those required by simple diffusion in an aqueous system. (g) Neither the inward or outward movement of Pi is modulated by trans-phosphate. These latter findings suggest that transport of phosphate across the human erythrocyte is compatible with slow facilitated diffusion with symmetry for influex and efflux. The transmembrane chemical distribution ratio, and the equilibrium flux of Pi were not different from normal in the X-LH erythrocyte. Nor did the extracellular Pi concentration, arsenate, or temperature affect Pi entry differently in the two types of cells. We dedjce that different gene products serve the diffusional type of Pi transport in the erythrocyte membrane and the saturable component of transepithelial absorption in the gut and kidney. Only the latter is affected by the X-LH mutation. The former is apparently present not only in erythrocytes but also in epithelial tissue, where it can serve the absorption of pharmacologic amounts of Pi in the therapeutic repair of the depleted phosphate pools in X-LH.

Amides↗
PubMed2001

The influence of depletion of voltage dependent anion selective channel on protein import into the yeast Saccharomyces cerevisiae mitochondria.

The supply of substrates to the respiratory chain as well as of other metabolites (e.g. ATP) into inner compartments of mitochondria is crucial to preprotein import into these organelles. Transport of the compounds across the outer mitochondrial membrane is enabled by mitochondrial porin, also known as the voltage-dependent anion-selective channel (VDAC). Our previous studies led to the conclusion that the transport of metabolites through the outer membrane of the yeast Saccharomyces cerevisiae mitochondria missing VDAC (now termed YVDAC1) is considerably restricted. Therefore we expected that depletion of YVDAC1 should also hamper protein import into the mutant mitochondria. We report here that YVDAC1-depleted mitochondria are able to import a fusion protein termed pSu9-DHFR in the amount comparable to that of wild type mitochondria, although over a considerably longer time. The rate of import of the fusion protein into YVDAC1-depleted mitochondria is dis- tinctly lower than into wild type mitochondria probably due to restricted ATP access to the intermembrane space and is additionally influenced by the way the supporting respiratory substrates are transported through the outer membrane. In the presence of ethanol, diffusing freely through lipid membranes, YVDAC1-depleted mitochondria are able to import the fusion protein at a higher rate than in the presence of external NADH which is, like ATP, transported through the outer membrane by facilitated diffusion. It has been shown that transport of external NADH across the outer membrane of YVDAC1-depleted mitochondria is supported by the protein import machinery, i.e. the TOM complex (Kmita & Budzińska, 2000, Biochim. Biophys. Acta 1509, 86-94.). Since the TOM complex might also contribute to the permeability of the membrane to ATP, it seems possible that external NADH and ATP as well as the imported preprotein could compete with one another for the passage through the outer membrane in YVDAC1-depleted mitochondria.

Adenosine Triphosphate↗
PubMed1998

The embryoid body as a novel in vitro assay system for antiangiogenic agents.

Tumor progression necessitates the induction of blood vessels that converge upon the tumor and enhance the diffusibility of oxygen and nutrients. Approaches to treat cancer by antiangiogenic therapy are therefore straightforward, and there is a great need for suitable in vitro systems to test antiangiogenic agents. In the present study, embryoid bodies (EBs) differentiated from totipotent mouse embryonic stem (ES) cells and cultivated using the spinner flask technique are introduced as an in vitro system for antiangiogenesis research. ES cells effectively differentiated endothelial cells within the three-dimensional tissue of EBs. The total area of capillary-like structures, which were positive for CD31 (platelet endothelial cell adhesion molecule, PECAM-1), was assessed by confocal laser scanning microscopy and image analysis of a series of optical sections. Endothelial differentiation occurred between Day 4-5 and Day 8 of EB development. Within 7 days, 100% of EBs contained capillary-like structures. Suramin, tamoxifen, tetrahydrocortisol, and a combination of tetrahydrocortisol and heparin were tested for their antiangiogenic capacity in the EB system and were found to efficiently inhibit endothelial differentiation. Diffusion studies of a 10-kd 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran and the fluorescent, amphiphilic agent doxorubicin in avascular and vascularized EBs revealed that the endothelial structures formed functional vessels that facilitated diffusion. The diffusion coefficient D for doxorubicin was 296 x 10(-9) cm2 s(-1) in vascularized 8-day-old EBs, ie, about 10-fold larger than in avascular 3-day-old EBs (18 x 10(-9) cm2 s(-1)) and EBs treated with suramin (14 x 10(-9) cm2 s(-1)), tamoxifen (13.5 x 10(-9) cm2 s(-1)), and tetrahydrocortisol/heparin (18.5 x 10(-9) cm2 s(-1)). Consequently, avascular EBs treated with antiangiogenic agents developed central necrosis, which was absent in vascularized EBs. Our findings indicate that EBs are a suitable in vitro model system to study the effects of antiangiogenic agents in a three-dimensional tissue context. Furthermore, EBs provide a unique model to investigate the diffusion of anticancer agents in a tissue in both the avascular and vascularized states.

Animals↗
PubMed2006

[Structural-functional analysis of diffusion in glucose absorption by rat small intestine enterocytes].

To elucidate mechanisms providing transport of sugars across intestinal epithelium, on taking into account the current hypotheses (active transport, participation of paracellular transport and passive component of transcellular transport), it was important to reveal structural changes of tight junctions and distribution of the carriers of facilitated diffusion of GLUT2 and protein kinase C during absorption of glucose. On using confocal and electron microscopy, ultrastructural and immunocytochemical studies of enterocytes after perfusion of isolated rat small intestine fragment with 75 mM glucose (chronic experiment) have shown: 1) fluorescent labels of transporter GLUT2 and PKCbetaII are located in the apical area of enterocytes situated at the upper half of the villus. Antibodies against GLUT2, conjugated with gold, are revealed at the microvilli or apical membrane and in the area of terminal network; 2) no ultrastructural changes of the tight junction are detected on ultrathin sections and freeze--fracture replics. At the same time, fluorescent and gold labels against actin are concentrated in the vicinity of the lateral membrane in the tight junction area. The results obtained can serve a confirmation of a hypothesis that at high glucose concentrations GLUT2 participates in its transfer across the apical membrane.

Animals↗
PubMed1985

Contraluminal phosphate transport in the proximal tubule of the rat kidney.

In order to study the characteristics of contraluminal phosphate transport the stopped flow microperfusion technique [13] has been applied. By measuring the time-dependent decrease of interstitial 33Pi concentration at different starting concentrations a simple diffusion kinetics with a permeability coefficient of 7.5 +/- 1.0 X 10(-8) cm2 s-1 was found. Such a kinetic was so far only observed with 2-deoxy-D-glucose. This substance, however, is transported in addition by facilitated diffusion as was seen by paraaminohippurate, methylsuccinate and sulfate. The contraluminal transport of phosphate was inhibited by H2-DIDS (5 mmol/l). It was, however, not influenced by omission of Na+ from the perfusates, by addition of sulfate (150 mmol/l), methylsuccinate (50 mmol/l), arsenate (50 mmol/l), the Hg-compound mersalyl (5 mmol/l), high and low phosphate diet and pH changes between 6.0 and 8.0. The data indicate that phosphate, which is reabsorbed from the lumen by a Na+-dependent transport system, leaves the cell by a rather unspecific contraluminal diffusion pathway.

Animals↗
PubMed1987

Diffusion coefficients of proteins in carrier ampholyte versus immobiline gels.

The apparent diffusion coefficients of proteins in carrier ampholyte isoelectric focusing (CA-IEF) and in immobilized pH gradients (IPGs) are strongly dependent on the amount of buffering ions present in the system. However, whereas in CA-IEF increased levels of ampholytes facilitate diffusion, in IPGs they strongly quench it. It is concluded that a protein in an IPG matrix is isoelectric but not isoionic, in the sense that it forms a salt with the surrounding ions bound to the polyacrylamide matrix. This salt formation is beneficial as it greatly increases protein solubility at the pI. It is suggested that, when performing zymograms in situ, the IPG gel should contain at least twice the standard amount of Immobiline, so as to keep sharp enzyme bands even with prolonged incubation periods.

Animals↗
PubMed1976

Glycerol transport in human red cells.

The kinetics of 14C-glycerol exchange was studied in human red cells. Glycerol appeared to be transported by two mechanisms: (i) by facilitated diffusion with permeability depending on glycerol concentration, and (ii) by an unspecific pathway, presumably representing the diffusion of individual glycerol molecules through the membrane with permeability independent of glycerol concentration. The latter permeability was 8 X 10(-8) cm/s at 20 degrees C, it was independent of pH, and had an activation energy of 25 kcal/mol. The facilitated transport of glycerol was completely inhibited by Cu++, and the activation energy was low, about 10 kcal/mol. The transport system was competitively inhibited by H+, reacting with at least three hydrogen analogue, as well as dimethylsulfoxide (a hydrogen bonding molecule with no structural resemblance to glycerol), inhibited glycerol transport competitively. Steins "dimerizer hypothesis" was revised according to our findings. A kinetic scheme describing the reactions of a transport controlling site with glycerol is presented in the Appendix. It is demonstrated in the article that the scheme accounts for out experimental results.

Biological Transport↗
PubMed1995

Calculation of oxygen pressures in tissue with anisotropic capillary orientation. I. Two-dimensional analytical solution for arbitrary capillary characteristics.

In tissue with a distinct orientation of the oxygen supplying structures, the capillaries, a mathematical description of oxygen transport is feasible in terms of two-dimensional diffusion in a plane perpendicular to the capillaries. Muscle tissue is an example of a highly anisotropic tissue. With some additional simplifying assumptions, a solution can be constructed in terms of capillary sources for arbitrary capillary characteristics, in particular, capillary locations. The solution includes facilitated diffusion by myoglobin in the tissue. For homogeneous tissue, the solution becomes explicit allowing direct calculation of tissue oxygen pressure at any location in a field of simple geometry (circular, rectangular). Also, the size of the area into which each capillary distributes its oxygen, the oxygen supply area, is readily calculated.

Animals↗
PubMedDate not supplied

Placental transport of hexoses: a comparative study with antipyrine and amino acids.

A comparative study of the transplacental passage of some labelled hexoses, amino acids and antipyrine leads to the following conclusions: The maternal-fetal transfer of D-glucose, which amounted to 90 per cent of antipyrine, is very efficient. D-glucose, 3-O-methylglucose and 2-deoxyglucose, for which equivalent transfers were obtained, appear to share the same carrier system. L-glucose transport occurs by simple diffusion. 3-O-methylglucose did not accumulate in the placental tissue. Similarly, maternal and fetal concentrations of 2-deoxyglucose were nearly at equilibrium. It is concluded that the transport of D-glucose is not concentrative. These observations provide further evidence of a facilitated diffusion process for the transport of D-glucose across the human placental membrane.

Amino Acids↗
PubMed1993

Resistance to O2 diffusion in anemic red muscle: roles of flux density and cell PO2.

Normal and anemic dog gracilis muscles were compared at equal O2 uptake rates (VO2) to locate the principal site of resistance to diffusive O2 transport. Anemia halved the hematocrit and the number of red blood cells per square millimeter of muscle cross section. Flow doubled in anemia, and flow times arterial O2 content, PO2 of effluent blood, and O2 extraction per red blood cell were approximately the same as control. Nevertheless, intracellular PO2 was significantly lower in anemia. At any instant the aggregate red blood cell surface area for O2 release was about half normal. Because the flux (VO2) was the same as control, the driving force for diffusion from red blood cell to myocyte should have doubled. An estimate of the total driving force from red blood cell to mitochondria was greater in anemia. This increase was much less than a factor of 2 because lower intracellular PO2 increases myoglobin-facilitated diffusion, thus decreasing resistance inside the myocyte. The role of myoglobin and the coupling of convective to diffusive transport are discussed. We conclude that the principal resistance to O2 diffusion lies outside the myocyte.

Anemia↗