Facilitated Diffusion Mechanism Research Articles

Calbindin Independence of Calcium Transport in Developing Teeth Contradicts the Calcium Ferry Dogma

Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin(28kDa). Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin(28kDa)-null mutants. An unchanged rate of calcium transport was verified by measurements of (45)Ca incorporation into developing teeth in vivo. In enamel-forming cells, the absence of calbindin(28kDa) was not compensated by other cytosolic calcium-binding proteins as detectable by (45)Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.

Nucleocytoplasmic shuttling of JAZ, a new cargo protein for exportin-5.

Exportin-5 is a nuclear export receptor for certain classes of double-stranded RNA (dsRNA), including pre-micro-RNAs, viral hairpin RNAs, and some tRNAs. It can also export the RNA binding proteins ILF3 and elongation factor EF1A. However, the rules that determine which RNA binding proteins are exportin-5 cargoes remain unclear. JAZ possesses an unusual dsRNA binding domain consisting of multiple C2H2 zinc fingers. We found that JAZ binds to exportin-5 in a Ran-GTP- and dsRNA-dependent manner. Exportin-5 stimulates JAZ shuttling, and gene silencing of exportin-5 reduces shuttling. Recombinant exportin-5 also stimulates nuclear export of JAZ in permeabilized cells. JAZ also binds to ILF3, and surprisingly, this interaction is RNA independent, even though it requires the dsRNA binding domains of ILF3. Exportin-5, JAZ, and ILF3 can form a heteromeric complex with Ran-GTP and dsRNA, and JAZ increases ILF3 binding to exportin-5. JAZ does not contain a classical nuclear localization signal, and in digitonin-permeabilized cells, nuclear accumulation of JAZ does not require energy or cytosol. Nonetheless, low temperatures prevent JAZ import, suggesting that nuclear entry does not occur via simple diffusion. Together, these data suggest that JAZ is exported by exportin-5 but translocates back into nuclei by a facilitated diffusion mechanism.

High capacity xylose transport in PYCC 4715

Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (μmax=0.5 h−1, 30°C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (Km=0.2 mM, Vmax=7.5 mmol h−1 g−1) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K=50 mM, Vmax=11 mmol h−1 g−1) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems.

High capacity xylose transport in Candida intermedia PYCC 4715.

Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (mu(max)=0.5 h-1, 30 degrees C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (Km = 0.2 mM, Vmax = 7.5 mmol h-1 g-1) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K = 50 mM, Vmax = 11 mmol h-1 g-1) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems.

Changes in ion content and transport during development of embryonic rainbow trout

Wet mass and water content of four lots of whole eggs did not change throughout embryonic development of rainbow trout Oncorhynchus mykiss. Eggs in all four lots accumulated Na+. Eggs in lots 2 and 4 also accumulated Ca2+ and Cl‐, whereas eggs in lot 1 showed no significant change in Ca2+ or Cl‐ and eggs in lot 3 showed no change in Cl‐and a small loss of Ca2+. Although the Na+ content of embryonic tissues increases in the later stages of development, the yolk sac content remained constant, indicating uptake of Na+ from the environment. Na+ uptake by whole eggs was non‐saturable, consistent with diffusion of Na+ across the chorion into the perivitelline fluid. Na+ uptake in dechorionated embryos was saturable, as was Ca2+ uptake by both whole eggs and dechorionated embryos, consistent with active uptake or facilitated diffusion mechanisms at the surface of embryos. Very low Ca2+ uptake rates in dechorionated embryos suggest that the Ca2+ uptake mechanism is not fully developed until after hatching.

Detection of two distinct transporter systems for 2-deoxyglucose uptake by the opportunistic pathogen Pneumocystis carinii

Since the opportunistic pathogen Pneumocystis carinii grows only slowly in vitro, the mechanism of glucose uptake was investigated to better understand how the organism transports nutrients. Using the non-metabolizable analogue 2-deoxyglucose, two uptake systems were detected with Q 10 values of 2.12 and 2.09, respectively. One had a high affinity ( K m=67.5 μM) and the other a low affinity ( K m=5.99 mM) for 2-deoxyglucose uptake. Glucose or deoxyglucose phosphate products from transported radiolabeled substrates were not detected during the incubation times used in this study. Both systems were inhibited by mannose, galactose, fructose, galactosamine, glucosamine, and glucose but not by allose, 5-thioglucose, xylose, glucose 6-phosphate and glucuronic acid. Salicylhydroxamate, KCN, iodoacetate, and 2,4-dinitrophenol inhibited the high-affinity transporter, suggesting it required ATP. Ouabain, monensin, carbonyl cyanide m-chlorophenylhydrazone, and N, N′-dicyclohexylcarbodiimide also inhibited deoxyglucose uptake, as did the replacement of Na + in the incubation medium with choline, indicating requirements for Na + and H +. The high-affinity system was also inhibited by the protein synthesis inhibitors cycloheximide and chloramphenicol. In contrast, the low-affinity system transported deoxyglucose by facilitated diffusion mechanisms. Unlike the human erythrocyte glucose transporter GLUT1, the P. carinii transporters recognized fructose and galactose and were relatively insensitive to cytochalasin B, suggesting that the P. carinii glucose transporters may be good drug targets.

Evidence for existence of a nuclear pore complex-mediated, cytosol-independent pathway of nuclear translocation of ERK MAP kinase in permeabilized cells.

The classical mitogen-activated protein kinase (MAPK, also known as ERK) pathway is widely involved in eukaryotic signal transductions. In response to extracellular stimuli, MAPK becomes activated and translocates from the cytoplasm to the nucleus. At least two pathways for the nuclear import of MAPK are shown to exist; passive diffusion of a monomer and Ran-dependent active transport of a dimer, the detailed molecular mechanism of which is unknown. In this study, we have reconstituted nuclear import of MAPK in vitro by using digitonin-permeabilized cells with GFP-fused MAPK protein (GFP-MAPK), which is too large to pass through the nuclear pore by passive diffusion. GFP-MAPK was able to accumulate in the nucleus irrespective of its phosphorylation state. This import of GFP-MAPK occurred even in the absence of any soluble cytosolic factors or ATP but was inhibited by wheat germ agglutinin or an excess amount of importin-beta or at low temperatures. Moreover, MAPK directly bound to an FG repeat region of nucleoporin CAN/Nup214 in vitro. Taken together, these results suggest the third pathway for nuclear import of MAPK, in which MAPK passes through the nuclear pore by directly interacting with the nuclear pore complex.

Assays of glucose entry, glucose transporter amount, and translocation.

Glucose enters the cell by a carrier-mediated, facilitated diffusion mechanism, which, in most tissues, exhibits no energy or counter-ion requirements. In adipose tissues and skeletal muscle, glucose entry is acutely regulated by insulin and other hormones (,). Indeed, in those tissues, glucose transporter 4 (GLUT4) is the chief isoform which is, in basal conditions, retained in a specific intracellular storage compartment (). The GLUT4-containing vesicles are translocated to the plasma membrane in response to insulin, thus allowing for the massive entry of glucose into the cells (,). Adipocytes also contain a small proportion of the ubiquituously expressed glucose transporter, GLUT1, which is at a similar level at the plasma membranes and inside the cell (). Because of this basal distribution, insulin effect on GLUT1 translocation is minor.

Synthesis, Lipophilicity, and Anti-HIV Activity of a New Brominated Analog of Zidovudine

A novel cyclic bromine zidovudine analog, (-)-trans-(5S,6S)-5-bromo-6,5′-epoxy-5,6-dihydro-3′-azido-3′-deoxythymidine (2), and its diastereoisomer (+)-trans-(5R,6R)-(3) were synthesized and characterized by spectroscopic methods, obtaining 3 in very low yields. The major product 2 presents a selectivity index (CCID50/IC50) similar to zidovudine but 55.5 times with higher lipophilicity, which should increase the ability of 2 to cross the blood-brain barrier by a non facilitated diffusion mechanism.

Mannitol transport by vacuoles of storage parenchyma of celery petioles operates by facilitated diffusion

Summary The sugar alcohol mannitol is the main water-soluble carbohydrate of the storage parenchyma of celery petioles. Compartmentation studies had indicated that mannitol was about equally distributed between the large central vacuole and the cytosol of these cells (Keller, F. and P. Matile, New Phytol., 113, 291–299 [1989]). Evidence is now presented for a tonoplast-bound mannitol carrier that operates by facilitated diffusion, i.e. protein-mediated and passively. With isolated parenchyma vacuoles it was shown that the uptake rate of mannitol, at 1 mmol/L external concentration, was about twice that of L-glucose and glucitol (sorbitol), both established markers for simple diffusion. Mannitol uptake was a saturable process with apparent K m and V max values of 14 mmol/L and 0.067 nmol/μL vacuoles · min, respectively. It was also quite specific as indicated by competition studies with glucitol, galactitol, glucose, and fructose. The functionality of the mannitol carrier was unaffected by three protein-modifying reagents, the sulfhydryl group modifiers p -chloro mercuribenzenesulfonic acid (PCMBS) and N -ethylmaleimide (NEM) and the histidine modifier diethyl pyrocarbonate (DEPC). Mannitol uptake was neither stimulated by energization with ATP or pyrophosphate nor was it impaired by dissipation of the pH gradient or the membrane potential and is, therefore, passive. The implications of this facilitated diffusion mechanism for the proposed physiological functions of mannitol in celery are discussed.

Transport of ascorbate into plasma membrane vesicles ofPhaseolus vulgaris L.

Incubation of bean hook plasma membrane vesicles in the presence of L-[14C]ascorbate (ASC) resulted in a specific recovery of significant levels of the ligand with the vesicles. The strong decrease in radioactive ASC detected after hypotonic disruption of the vesicles or after an assay at 4 °C indicated that ASC was probably transported from the medium into the lumen of the membrane vesicles. The concentration kinetics of this presumptive transport process revealed a saturation curve which best fitted a biphasic model. Each phase in this model showed Michaelis-Menten type kinetics. The kinetic parameters for the different phases were calculated to be 14 and 79 μM (Km1 andKm2) and 26 and 53 pmol/min · mg protein (Vmax1 andVmax2). High concentrations of iso-ascorbate, dehydroascorbate (DHA) or non-labelled ASC significantly reduced the uptake of the radioactive vitamin. It was demonstrated that sugar or amino acid carriers are not involved in the ASC transport reaction. Generation of transmembrane cation gradients (H+, K+, Ca2+, Na+) or addition of sulfhydryl reagents (pCMBS or NEM) did not affect the ASC uptake in any way. It is suggested that ASC is taken up by a facilitated diffusion mechanism.

Inducer expulsion is not a determinant of diauxic growth in Streptococcus bovis.

When Streptococcus bovis JB1 was repeatedly transferred in a medium that contained the non-metabolizable glucose analog, 2-deoxyglucose, it lost its phosphotransferase system (PTS) for glucose but was still able to take up glucose via a facilitated diffusion mechanism. The wild type (JB1) had an inducible enzyme II lactose, but the mutant (JB1(2DG)) had a constitutive lactose PTS. JB1(2DG) was no longer able to exclude lactose when it was provided with glucose, but it retained its ability to expel a non-metabolizable lactose analog. Because JB1(2DG) could utilize glucose and lactose simultaneously and grow in a non-diauxic fashion, it appeared that inducer expulsion was not an important catabolite regulatory mechanism. Based on these results, inducer expulsion may be an artifact of non-metabolizable sugars.

Facilitated diffusion of the EcoRI DNA methyltransferase is described by a novel mechanism.

The contribution of nonspecific DNA to binding parameters (K(d), k(off), and k(on)) was determined for the EcoRI DNA methyltransferase under noncatalytic conditions. An increase in DNA size from 14 to 775 base pairs causes a 20-fold decrease in K(d), while k(off) remains constant over the same range. The calculated k(on) increases with longer substrates, consistent with a facilitated diffusion mechanism. However, the combined results deviate from the model developed to describe facilitated diffusion [Berg, O. G., Winter, R. B., & von Hippel, P. H. (1981) Biochemistry 20, 6929-6948]. Our results were successfully simulated using numerical integration of a kinetic scheme invoking protein dissociation via the ends of DNA. Consistent with this scheme, the methyltransferase dissociates more slowly from a circularized DNA molecule than from the identical linearized form. The simulation strategy correctly models our data with the methyltransferase and should be generally useful for routine modeling of facilitated diffusion involving protein-DNA systems.

Accurate Scanning of the BssHII Endonuclease in Search for Its DNA Cleavage Site

A facilitated diffusion mechanism has been proposed to account for the kinetic efficiency with which restriction endonucleases are able to locate DNA recognition sites. Such a mechanism involves the initial formation of a nonspecific complex upon collision of the protein with the DNA, with the subsequent diffusion of the protein along the DNA helix until either a recognition site is located or the protein dissociates into solution. Protein translocation may be facilitated by either sliding along the DNA, hopping to nearby sites, or intersegment transfer over larger distances. Previous analyses of the manner in which restriction enzymes cleave DNA substrates did rule out the latter mechanism. To discriminate between protein sliding or scanning and protein hopping, we designed a unique DNA template with three overlapping, mutually exclusive recognition sites for the BssHII endonuclease. Analysis of the cleavage pattern demonstrated efficient usage of both external sites, whereas the centrally located site was not efficiently cleaved. These results confirm that linear diffusion of the BssHII enzyme occurs by scanning along the DNA. Furthermore, the scanning enzyme was found to stop and cleave at the first site encountered. Thus, a sliding restriction endonuclease recognizes cleavage sites with high fidelity, without skipping of potential sites.

Contribution of facilitated diffusion and processive catalysis to enzyme efficiency: implications for the EcoRI restriction-modification system.

The contribution of nonspecific DNA to enzyme efficiency (k(cat)/K(m)) is described for a sequence-specific DNA-modifying enzyme. Our investigation focuses on the EcoRI DNA methyltransferase which transfers a methyl group from the cofactor S-adenosylmethionine to the second adenine in the double-stranded DNA sequence GAATTC. k(cat)/K(m) increases 4-fold as DNA length increases from 14 to 429 base pairs and increases 2-fold as the distance from the site to the nearest end is increased from 29 to 378 base pairs. No changes in k(cat)/K(m) result from further increases in either case. A facilitated diffusion mechanism is proposed in which the methyltransferase scans an average of <400 base pairs prior to dissociation from a DNA molecule. The methyltransferase was found to methylate two sites on a single DNA molecule in a distributive rather than a processive manner, suggesting that the enzyme dissociates from the DNA prior to release of the reaction product S-adenosylhomocysteine. A direct competition experiment with the EcoRI endonuclease shows the methyltransferase to be slightly more efficient at specific site location and catalysis. A rationale for the role of facilitated diffusion in this type II restriction-modification system is proposed.

Alternative strategies of 2-deoxyglucose resistance and low affinity glucose transport in the ruminal bacteria, Streptococcus bovis and Selenomonas ruminantium

Streptococcus bovis and Selenomonas ruminantium grew in the presence of the glucose analog, 2-deoxyglucose (2-DG), but the cells no longer had high affinity glucose transport. In S. bovis, 2-DG resistance was correlated with a decrease in phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase (PTS) activity. The 2-DG-selected S. bovis cells relied solely upon a low affinity, facilitated diffusion mechanism of glucose transport and a 2-DG-resistant glucokinase (ATP-dependent). The glucokinase activity of S. ruminantium was competitively inhibited by 2-DG, and the 2-DG selected cells continued to use PEP-dependent PTS as a mechanism of glucose transport. In this latter case, the 2-DG selected cells switched from a mannosephosphotransferase (enzyme II) that phosphorylated glucose, mannose, and 2-DG, but not α-methylglucoside to a glucosephosphotransferase (enzyme II) that phosphorylated glucose and α-methylglucoside but not 2-DG or mannose. The glucosephosphotransferase (enzyme II) had a very low affinity for glucose and the transport kinetics were similar to the facilitated diffusion system of S. bovis.

Alternative strategies of 2-deoxyglucose resistance and low affinity glucose transport in the ruminal bacteria, Streptococcus bovis and Selenomonas ruminantium.

Streptococcus bovis and Selenomonas ruminantium grew in the presence of the glucose analog, 2-deoxyglucose (2-DG), but the cells no longer had high affinity glucose transport. In S. bovis, 2-DG resistance was correlated with a decrease in phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase (PTS) activity. The 2-DG-selected S. bovis cells relied solely upon a low affinity, facilitated diffusion mechanism of glucose transport and a 2-DG-resistant glucokinase (ATP-dependent). The glucokinase activity of S. ruminantium was competitively inhibited by 2-DG, and the 2-DG selected cells continued to use PEP-dependent PTS as a mechanism of glucose transport. In this latter case, the 2-DG selected cells switched from a mannosephosphotransferase (enzyme II) that phosphorylated glucose, mannose, and 2-DG, but not alpha-methylglucose to a glucosephosphotransferase (enzyme II) that phosphorylated glucose and alpha-methylglucoside but not 2-DG or mannose. The glucosephosphotransferase (enzyme II) had a very low affinity for glucose and the transport kinetics were similar to the facilitated diffusion system of S. bovis.

Adenosine transport in cultured human umbilical vein endothelial cells is reduced in diabetes.

Adenosine transport in cultured human umbilical vein endothelial cells (HUVEC) was characterized and shown to be mediated by a single facilitated diffusion mechanism. Initial rates of adenosine influx at 22 degrees C were saturable [apparent Michaelis constant, 69 +/- 10 microM; maximum velocity (Vmax), 600 +/- 70 pmol.10(6) cells-1.s-1] and inhibited by nitrobenzylthioinosine (NBMPR). Formycin B had an unusually high affinity [inhibitory constant (Ki), 18 +/- 4.3 microM], whereas inosine had a low affinity (Ki, 440 +/- 68 microM) and nucleobases were without effect on adenosine influx. The number of transporters (1.2 x 10(6) sites/cell) was estimated by NBMPR equilibrium binding (apparent dissociation constant, 0.11 +/- 0.01 nM; maximum binding, 2.0 +/- 0.15 pmol/10(6) cells). In addition, we compared these endothelial cells with those obtained from cords from pregnancies complicated by diabetes (HUVEC-D), since embriopathy may occur in these conditions. HUVEC-D exhibited a 2.3-fold reduction in both the Vmax for adenosine influx and the maximum number of NBMPR binding sites (260 +/- 40 pmol.10(6) cells-1.s-1 and 0.86 +/- 0.08 pmol/10(6) cells, respectively). However, the turnover number for each nucleoside transporter in normal and diabetic HUVEC was similar (approximately 300 adenosine molecules/s). Adenosine metabolism at 10 microM in HUVEC-D was modified compared with normal cells. Intracellular phosphorylation (> 90%) was the predominant pathway in normal HUVEC, whereas in HUVEC-D, substantial levels of adenine and adenosine were detected. The present results demonstrate therefore the downregulation of the NBMPR-sensitive nucleoside transporter and changes in adenosine metabolism in HUVEC from diabetic pregnancies.

Carrier-mediated transport of H1-antagonist at the blood-brain barrier: mepyramine uptake into bovine brain capillary endothelial cells in primary monolayer cultures.

The transport mechanism of the H1-antagonist mepyramine at the blood-brain barrier (BBB) was studied by using primary cultured monolayers of bovine brain capillary endothelial cells (BCEC). The initial uptake of [3H]mepyramine into the BCEC showed strong temperature and concentration dependency, indicating that it involves both saturable and nonsaturable processes. Transport at the luminal membrane may be the rate-limiting process in the transcellular transport, since the values of the uptake coefficient of [3H]mepyramine at the luminal membrane (609 microliters/mg protein/min) and the transcellular permeability coefficient (488 microliters/mg protein/min) are very similar. The initial uptake of [3H]mepyramine was not affected by metabolic inhibitors, but was stimulated by preloading with the drug. Mepyramine appears to be transported into the BCEC by a carrier-mediated transport system which does not require metabolic energy, probably via a facilitated diffusion mechanism.

Membrane extraction of mercury (II) and silver (I) by bis(di(2-ethylhexyloxy)thiophosphoryl)disulfide

A supported liquid membrane containing bis(di(2-ethylhexyloxy)thiophosphoryl)disulfide (BTPD) as a novel membrane carrier was prepared and tested. Metal ion transfer was shown to be accelerated by the presence of thiourea or thiosulfate in the receiver stripping solutions. Under varying conditions, mercury and silver transfer rates of up to 7 and 3 μmol/m 2s, were measured. Mercury transport mechanism was demonstrated to be dependent on the nature of the anion. Mercury nitrate transport appears to be mainly due to a coupled Hg 2+H + facilitated diffusion mechanism, according to the ion-exchange stoichiometry. Mercury halides were transported mainly as neutral complexed species due to carrier-facilitated diffusion. Selectivity of the membrane for mercury and silver over zinc, nickel, cadmium and copper has been found to be of the order of 10 3.