Diffusion, chemical reaction and facilitated diffusion.
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Blood Proteins↗
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Studies of the beta-galactoside transporter in inverted membrane vesicles of Escherichia coli. I. Symmetrical facilitated diffusion and proton gradient-coupled transport.
Facilitated diffusion of [14C]lactose into inverted membrane vesicles of Escherichia coli was measured using HgCl2 as a stopping reagent and polylysine to flocculate the vesicles for filtration. Equilibration of lactose between the internal and external volumes required expression of the y gene of the lac operon and was inhibited by thiodigalactoside or by prior incubation with N-ethylmaleimde or HgCl2. The initial rate of uptake was saturable, with a Kt of 0.95 mM. Counterflow of [14C]lactose was demonstrated in either direction. ATP hydrolysis or respiration drove the efflux of internal lactose. The effect of ATP required addition of F1 coupling factor (ATPase) from E. coli when lactose transport was studied in F1-deficient inverted vesicles. Accumulation of lactose against a concentration gradient was achieved by forming an artificial electrochemical proton gradient consisting of a membrane potential negative inside or a pH gradient basic inside. Addition of ATP inhibited this proton driven uptake showing that it occurred in inverted vesicles. It was concluded that the lactose-proton co-transport protein (M protein) is qualitatively symmetrical with respect to the facilitated diffusion of lactose and the coupling of proton and lactose transport.
Biological Transport↗
Theory of the rotational contribution to facilitated diffusion.
Gros and others have recently shown experimentally that the facilitated diffusion of protons carried by a form of haemoglobin is enhanced by rotational diffusion of the carrier, whereas facilitated diffusion of oxygen by the same carrier is not. In this paper the theory of facilitated transport by rotating carriers is developed from first principles. The theory confirms Gros's findings that (i) the rotational contribution appears only when the angle of rotational diffusion over the average time the proton remains bound is small and (ii) under these conditions rotation enhances the normal translational contribution by a factor 1/2 at the lowest carrier concentrations. The theory also shows that there must be a rotational boundary layer.
Binding Sites↗
Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair.
Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.
Biological Transport↗
Influence of membrane lipid fluidity on glucose and uridine facilitated diffusion in human erythrocytes.
A central question which must be resolved before acceptable molecular descriptions of facilitated diffusion systems can be provided is the nature of the spatial and functional relationships between the transport proteins and the membrane lipids. In the work reported here, this question was addressed by investigating the dependence of the rates of glucose and uridine facilitated diffusion in human erythrocytes on membrane lipid fluidity. Two approaches were used to alter the lipid fluidity: treatment with ether, an anesthetic, and the exchange of a synthetic 3-ketosteroid, cholest-4-en-3-one, for membrane chloesterol. Both of these treatments result in a significant increase in membrane lipid fluidity, as judged by the increase in the rates of passive diffusion of uridine through cell membranes and of glucose through membrane lipid bilayer vesicles. Ether produces no change in the Km of either transport process, a slight decrease in the V for glucose transport, and no significant change in the V for uridine transport. Replacement of membrane cholesterol by cholest-4-en-3-one reduces the V for glucose transport slightly, without altering the Km, and reduces both the Km and V for uridine transport. The absence of the expected increase in the V of facilitated diffusion with increasing membrane lipid fluidity observed here with human erythrocytes is not consistent with models for the transport process which feature movement of transport proteins which are in direct contact with the bulk lipids of the membrane.
Biological Transport↗
Structure and regulation of the glpFK operon encoding glycerol diffusion facilitator and glycerol kinase of Escherichia coli K-12.
The glpFK operon maps near minute 88 on the linkage map of Escherichia coli K-12 with glpF promoter proximal. The glpF gene encodes a cytoplasmic membrane protein which facilitates the diffusion of glycerol into the cell. The glpK gene encodes glycerol kinase. In the present work, the nucleotide sequence of the 5'-end of the operon, including the control region, the glpF gene, and part of the glpK gene, was determined. The facilitator was predicted to contain 281 amino acids with a calculated molecular weight of 29,780. It is a highly hydrophobic protein with a minimum of six potential transmembrane alpha helices. The transcription start site for the glpFK operon was located 71 base pairs upstream from the proposed translation start codon for glpF. Preceding the transcription start site were sequences similar to the -10 and -35 consensus sequences for bacterial promoters. Binding sites for the cAMP-cAMP receptor protein (CRP) complex and the glp repressor were identified by DNase I footprinting. The region protected by the cAMP.CRP complex contained tandem sequences resembling the consensus sequence for CRP binding. The CRP sites were centered at 37.5 and 60.5 base pairs upstream of the start of transcription. The glp repressor protected an extensive area (-89 to -7 relative to the start point of transcription), sufficient for the binding of four repressor tetramers. Two additional binding sites for the repressor were identified within the glpK coding region. The DNA containing these two operators synergistically increased the apparent affinity of glp repressor for DNA fragments containing the four operators in the promoter region of the glpFK operon. With this study, a total of 13 operators for the glp regulon have been characterized. Comparison of these operators revealed the consensus 5'-WATGTTCGWT-3' for the operator half-site (W = A or T). The relative affinity of the glp repressor for the various glp operators was assessed in vivo using a promoter-probe vector. The relative apparent affinity of the control regions for glp repressor was glpFK greater than glpD greater than glpACB greater than glpTQ. The degree of catabolite repression for each of the operons was assessed using a similar system. In this case, the relative sensitivity of the glp operons to catabolite repression was glpTQ greater than glpFK greater than glpACB greater than glpD.
Amino Acid Sequence↗
Facilitated diffusion in a tissue cylinder with an anoxic region.
When the diffusion of oxygen is facilitated by myoglobin this diffusional transport can adequately supply oxygen required by tissue metabolism over greatly increased distances. Earlier analyses of this phenomenon have considered the rate of reaction between oxygen and myoglobin as infinitely fast. A first-order correction which takes into account the finite rate of reaction involves a reaction boundary layer at the surface where the oxygen enters the tissue, and a second reaction boundary layer around an anoxic region in the tissue when such a region is present. These corrections are calculated for a tissue cylinder and the results obtained are compared with results neglecting the finiteness of the reaction rate. The maximum distance over which facilitated diffusion can supply oxygen is less when the corrections are taken into account. For physiologically relevant parameter values the size of an anoxic region is predicted to be larger by about 15% of the radius of the tissue cylinder.
Biological Transport↗
Facilitated diffusion of CO2 across albumin solutions.
The steady-state CO(2) flux across thin layers of 30 g/100 ml albumin solutions was measured in two different CO(2) partial pressure ranges (boundary PCO(2) values 3 and 8 torr, and 160 and 650 torr, respectively). From the data the apparent diffusion coefficient for CO(2), DCO(2), was calculated. In the high PCO(2) range a value of DCO(2) was found which is to be expected on the basis of diffusion of dissolved CO(2) only. In the low PCO(2) range DCO(2) was about 100% higher than in the high PCO(2) range, when carbonic anhydrase was present and the pH was approximately 7.7. DCO(2) depended on the concentration of carbonic anhydrase. It increased with increasing pH. It is concluded that an additional diffusion of bound CO(2) (facilitated CO(2) diffusion) occurs in the low PCO(2) range and that this transport involves the hydration of CO(2). From the diffusion coefficients in the two PCO(2) ranges the rate of facilitated diffusion was determined. Approximate calculations show that this rate (at pH </= 7.7) can be explained on the basis of the proposed mechanism of facilitated CO(2) diffusion: bicarbonate diffusion and simultaneous proton transport by albumin diffusion. The view that facilitated CO(2) diffusion is mediated by the diffusion of albumin is supported by the experimental finding of a considerable suppression of the facilitated CO(2) flux in the presence of gelatinized agar-agar.
Acetazolamide↗
Facilitated diffusion of calcium across the rat intestinal epithelial cell.
Similarities and differences between the models for facilitated diffusion of substrates across cytoplasm and across cell membranes are discussed. It is valuable to consider that calbindin acts to increase the partitioning of calcium within the cytoplasm. Calculations on the basis of a simple model for this facilitated diffusion show that the rate of transcellular calcium movement depends in a complex way on KCAB, the dissociation constant for calcium and calbindin. At each value of the transcellular concentration gradient of free calcium, there is an optimal value of KCAB that gives the maximal augmentation of calcium flow. For low values of the ratio of free calcium concentrations at the opposite poles of the cell, the optimal value of KCAB is close to the prevailing concentration of free calcium. An analysis of possible effects of pH on binding of calcium to calbindin shows that it may be useful to consider cotransport of calcium and protons within the cell. Small values of a transcellular pH gradient can have large effects on enhancing transcellular calcium flow, provided the hydrogen ion concentration affects the binding of calcium to calbindin or the rate of diffusion of the calcium/calbindin complex.
Animals↗
Facilitated diffusion of a DNA binding protein on chromatin.
Facilitated diffusion accounts for the rapid rate of association of many bacterial DNA binding proteins with specific DNA sequences in vitro. In this mechanism the proteins bind at random to non-specific sites on the DAN and diffuse (by 'sliding' or 'hopping') along the DNA chain until they arrive at their specific functional sites. We have investigated whether such a mechanism can operate in chromatin by using a bacterial DNA binding protein, Escherichia coli RNA polymerase, that depends on linear diffusion to locate initiation sites on DNA. We have measured the competition between chromatin and its free DNA for the formation of initiation complexes. Only the short linker segments exposed by the removal of histone H1 are available for interaction with the polymerase, but the sparsely distributed promoter sites on the linker DNA of such a polynucleosome chain are located at the same rate as those on DNA. We conclude that the polymerase is free to migrate between the separate linker DNA segments of a polynucleosome chain to reach a promoter site. This chain thus permits the 'hopping' of proteins between neighboring linker segments in their search for a target site on the accessible DNA.
Animals↗
Melibiose permease of Escherichia coli. Characteristics of co-substrates release during facilitated diffusion reactions.
The mechanism of melibiose symport by the melibiose permease of Escherichia coli was investigated by further analyzing the Na+ (H+ or Li+)-coupled facilitated diffusion reactions catalyzed by the carrier in de-energized membrane vesicles, with particular emphasis on the reaction of sugar exchange at equilibrium. It is first shown that melibiose exchange at equilibrium proceeds without concomitant movement of Na+, i.e. the coupled cation is kinetically occluded during the melibiose exchange reaction. These results provide further experimental support for the model of Na+ sugar co-transport of the physiological substrate melibiose previously suggested (Bassilana, M., Pourcher, T., and Leblanc, G. (1987) J. Biol. Chem. 262, 16865-16870) in which: 1) the mechanisms of co-substrate binding to (or release from) the carrier are ordered processes on both the outer (Na+ first, sugar last) and inner membrane surfaces (sugar first, Na+ last) and give rise to a mirror-type model; 2) release of Na+ from the carrier on the inner membrane surface is very slow and rate-limiting for carrier cycling but is fast on the opposite side, contributing to the asymmetrical functioning of the permease. On the other hand, analysis of the exchange of identical sugars (homologous exchange) and different sugar analogs (heterologous exchange) indicates that the overall rate of sugar exchange reaction coupled to Na+ or Li+ is limited by the rate of one (or more) partial step(s) associated with the inflow of co-substrates and most probably by the rate of sugar release into the intravesicular medium. It is proposed that the variability of the facilitated diffusion reactions catalyzed by the carrier in the presence of different coupled cations and/or sugar analogs reflects variations in the rate of co-substrate release from the carrier on the inner membrane surface.
Cations, Monovalent↗
The permeability of rat liver lysosomes to sugars. Evidence for carrier-mediated facilitated diffusion.
1. By the osmotic-protection method, the penetration of sugars through the rat liver lysosomal membranes was studied with a view of determining whether sugar uptake was by facilitated diffusion. 2. The following criteria for this type of transport were established: sugar specificity, the order of uptake being 2-deoxy-D-glucose less than D-glucose less than D-mannose less than D-galactose less than D-ribose less than 2-deoxy-D-ribose; stereospecificity, the uptake of L-glucose and L-ribose being 50% slower than their D-stereoisomers; inhibition by 1 MM-phlorrhizin and 1 M-cytochalastin B; competition between sugars for uptake, and a Q10 (rate difference over a 10 degrees C temperature range) for uptake of approx. 2.8. 3. It is proposed that sugar uptake into lysosomes from rat liver is by facilitated diffusion.
4-Nitrophenylphosphatase↗
Pyruvate transport across the plasma membrane of the bloodstream form of Trypanosoma brucei is mediated by a facilitated diffusion carrier.
The characteristics of pyruvate transport across the plasma membrane in the bloodstream form of Trypanosoma brucei were studied using [14C]pyruvate in combination with the silicone-oil centrifugation technique. We present evidence for the existence of a facilitated diffusion carrier in the plasma membrane of T. brucei which specifically mediates the translocation of pyruvate. The uptake of pyruvate followed saturation kinetics (Km 1.96 +/- 0.28 mM; Cmax 36.61 +/- 1.15 nmol pyruvate/30 sec.mg protein), after correction of the data for a nonsaturable diffusion component. The uptake of pyruvate was competitively inhibited by a number of (oxo)monocarboxylic acids, including pyruvate analogs and metabolically related substances, but not by L-lactate. The transport exhibited the phenomenon of transacceleration, indicative for the involvement of a facilitated diffusion carrier. The carrier is highly specific for pyruvate and differs from other known monocarboxylate carriers present in the mitochondrial and/or plasma membrane of other eukaryotic cells in that it does not transport L-lactate.
Animals↗
Experimental analysis of ion/solute cotransport by substrate binding and facilitated diffusion.
An ion/solute cotransporter can be studied in the absence of a transmembrane gradient of the electrochemical potential of the ion. Inspection of the appropriate equations discloses that basic parameters of the cotransport cycle can be obtained by measuring cosubstrate binding and the initial-velocity kinetics of four modes of facilitated diffusion as a function of the concentration of the cotransported ion. The following information can be derived: estimates of the affinities of both cosubstrates, the number of binary intermediates participating in cotransport (equivalent to determining the order of cosubstrate binding and release), and the rate constants for the reorientation of the binding sites during cotransport. In general, both maximal velocities and half-saturation constants for the facilitated diffusion of one cosubstrate depend upon the concentration of the other. In some cases, the maximal velocities of influx and efflux do not increase monotonically with the concentration of the ion but pass through a maximum and decrease. If direct binding studies are not possible, affinities of the cosubstrates can be estimated from data for equilibrium exchange or countertransport. Also, an approximate description of the time course of the transient accumulation (overshoot) during countertransport is derived. Under certain circumstances, the height of the overshoot is proportional to the concentration of the cotransported ion. Thus, countertransport can be employed as a simple test to establish if a solute is cotransported with a particular ion. This treatment allows many effects noted in galactoside countertransport in Escherichia coli to be explained.
Biological Transport, Active↗
Transport and countertransport of thymidine in ATP depleted and thymidine kinase-deficient Novikoff rat hepatoma and mouse L cells: evidence of a high Km facilitated diffusion system with wide nucleoside specificity.
Incubation of cultured Novikoff rat hepatoma and mouse L cells in a glucose-free basal medium containing 5 mM KCN and 5 mM iodoacetate for about 10 minutes resulted in a complete depletion of the cells of ATP. ATP-depleted wild type cells or thymidine kinase-deficient sublines of Novikoff or L cells took up thymidine rapidly from the medium without concentrating it intracellularly, and exhibited countertransport of thymidine. Thus uptake was by facilitated diffusion. This transport system differs from the substrate-specific, low-Km (0.5 muM] thymidine transport system previously described for various types of cultured cells in that it exhibits an at least 100-fold higher Km and transports equally well various ribo- and deoxyribonucleosides. The results suggest that the rate-limiting step in thymidine incorporation into the nucleotide pool by wild type cells is phosphorylation rather than transport, or that the cells possess two transport systems, a facilitated diffusion system with low substrate specificity and a second system which involves substrate phosphorylation by thymidine kinase.
Adenosine Triphosphate↗
Facilitated diffusion during catalysis by EcoRI endonuclease. Nonspecific interactions in EcoRI catalysis.
The potential for processive EcoRI endonuclease hydrolysis has been examined on several DNA substrates containing two EcoRI sites which were embedded in identical sequence environments. With a 388-base pair circular DNA, in which the two recognition sites are separated by 51 base pairs (shorter distance) or 337 base pairs (longer distance), 77 and 34% of all events involved processive hydrolysis at ionic strengths of 0.059 and 0.13, respectively. However, the frequency of processive action on linear substrates, in which the two sites were separated by 51 base pairs, was only 42 and 17% at these ionic strengths, values half those observed with the circular DNA. Processive action was not detectable on circular or linear substrates at an ionic strength of 0.23. These findings indicate that DNA search by the endonuclease occurs by facilitated diffusion, a mechanism in which the protein locates and leaves its recognition sequence by interacting with nonspecific DNA sites. We suggest that processivity on linear substrates is limited to values half that for small circles due to partitioning of the enzyme between the two products generated by cleavage of a linear molecule. Given such topological effects, measured processivity values imply that the endonuclease can diffuse within a DNA domain to locate and recognize an EcoRI site 50 to 300 base pairs distant from an initial binding site, with minimum search efficiencies being 80 and 30% at ionic strengths of 0.059 and 0.13, respectively. The high efficiency of processive action indicates that a positionally correlated mode of search plays a major role in facilitated diffusion in this system under such conditions. Also consistent with this view was the identification of a striking position effect when two closely spaced EcoRI sites were asymmetrically positioned near the end of a linear DNA. The endonuclease displays a substantial preference for the more centrally located recognition sequence. This preference does not reflect differential sensitivity of the two sites to cleavage per se, but can be simply explained by preferential entry of the enzyme via the larger nonspecific target available to the more centrally positioned recognition sequence. These conclusions differ from those of a previous qualitative analysis of endonuclease processivity over short distances (Langowski, J., Alves, J., Pingoud, A., and Maass, G. (1983) Nucleic Acids Res. 11, 501-513).
Base Sequence↗
Facilitated diffusion and sodium-dependent transport of purine and pyrimidine nucleosides in rat liver.
In mammalian cells, nucleoside transport usually is mediated by facilitated diffusion. In addition, a Na(+)-dependent, concentrative nucleoside transport system has been detected in several tissues but not the liver. To further clarify hepatic nucleoside transport mechanisms, we measured the uptake of [2-14C]uridine (2 to 100 mumol/L) and of [8-14C]adenosine (10 to 75 mumol/L) by the isolated perfused rat liver in the presence or absence of extracellular sodium or specific inhibitors of facilitated nucleoside diffusion. Uridine transport and metabolism were monitored by the release of labeled catabolites including 14CO2, which indicated complete degradation of the pyrimidine. Adenosine, uridine and uridine catabolites were measured in the effluent perfusate by reversed-phase high-performance liquid chromatography and a radioactivity flow monitor. The existence of a Na(+)-dependent nucleoside transport system could be inferred from the following observations: (a) Sodium depletion caused a strong inhibition of nucleoside transport reflected by an up to threefold and 15-fold increase in extracellular uridine and adenosine, respectively. The sodium-dependent transport of uridine was saturated when the influent uridine concentration was raised beyond 20 mumol/L. No such saturation was observed for much higher concentrations of adenosine used (10 to 75 mumol/L). (b) Na(+)-free perfusion resulted in a strong suppression of the release of uridine catabolites by the liver. Complete uridine breakdown was depressed to 7% of the amount of 14CO2 released in the presence of sodium and at influent uridine concentrations below 20 mumol/L. (c) Inhibition of uridine (10 mumol/L) transport and degradation was observed after coperfusion with adenosine, deoxyadenosine, guanosine and deoxyguanosine.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Defective facilitated diffusion of nucleosides, a primary mechanism of resistance to 5-fluoro-2'-deoxyuridine in the HCT-8 human carcinoma line.
In vitro resistance of HCT-8 cells to 5-fluoro-2'-deoxyuridine (FdUrd) has been obtained after a stepwise increase (up to 1 microM) in the concentration of the nucleoside in the culture medium over a period of 6 months. With a clonogenic assay, the toxicities of 17 antineoplastic agents on HCT-8-sensitive and -resistant cells were compared. Resistant cells were 700-fold resistant to FdUrd and showed different degrees of cross-resistance to several purine and pyrimidine nucleoside analogues; no cross-resistance was noted to base analogues and other cytotoxic drugs. The activities of FdUrd phosphorylase, 5'-fluorouridine kinase, 5-fluorouridine phosphorylase, 5-fluorouracil phosphoribosyltransferase, and thymidylate synthase were not significantly different in the sensitive and resistant cell lines. Mixing experiments indirectly excluded the possible elevation of the level of cytoplasmic phosphatases. The activity of FdUrd kinase in sensitive cell extracts was no more than twice that of resistant cells, and the affinities of this enzyme for FdUrd and thymidine at 0.1 to 50 microM were similar in both cell lines. However, cultures of this line failed to accumulate 5-fluoro-2'-deoxyuridylate at concentrations of FdUrd that resulted in substantial accumulation of the nucleotide in the sensitive line. These contrasting data suggested a defect in the facilitated diffusion of the analogue. The entrance of free nucleoside and its subsequent phosphorylation were compared in the two lines over short (2 to 40 s) and longer time periods at 25 degrees C and at 4 degrees C over a range of extracellular FdUrd concentrations (0.1 to 10 microM). Rapid entrance of the nucleoside into sensitive cells was observed, but entry was not detectable in resistant cells. Dipyridamole and nitrobenzylthioinosine inhibition as well as high-performance liquid chromatography analysis confirmed that data obtained from the sensitive cell line during the first 40 s primarily reflected facilitated diffusion of free nucleoside.
Adenocarcinoma↗