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PubMed1990

Proton-linked sugar transport systems in bacteria.

The cell membranes of various bacteria contain proton-linked transport systems for D-xylose, L-arabinose, D-galactose, D-glucose, L-rhamnose, L-fucose, lactose, and melibiose. The melibiose transporter of E. coli is linked to both Na+ and H+ translocation. The substrate and inhibitor specificities of the monosaccharide transporters are described. By locating, cloning, and sequencing the genes encoding the sugar/H+ transporters in E. coli, the primary sequences of the transport proteins have been deduced. Those for xylose/H+, arabinose/H+, and galactose/H+ transport are homologous to each other. Furthermore, they are just as similar to the primary sequences of the following: glucose transport proteins found in a Cyanobacterium, yeast, alga, rat, mouse, and man; proteins for transport of galactose, lactose, or maltose in species of yeast; and to a developmentally regulated protein of Leishmania for which a function is not yet established. Some of these proteins catalyze facilitated diffusion of the sugar without cation transport. From the alignments of the homologous amino acid sequences, predictions of common structural features can be made: there are likely to be twelve membrane-spanning alpha-helices, possibly in two groups of six; there is a central hydrophilic region, probably comprised largely of alpha-helix; the highly conserved amino acid residues (40-50 out of 472-522 total) form discrete patterns or motifs throughout the proteins that are presumably critical for substrate recognition and the molecular mechanism of transport. Some of these features are found also in other transport proteins for citrate, tetracycline, lactose, or melibiose, the primary sequences of which are not similar to each other or to the homologous series of transporters. The glucose/Na+ transporter of rabbit and man is different in primary sequence to all the other sugar transporters characterized, but it is homologous to the proline/Na+ transporter of E. coli, and there is evidence for its structural similarity to glucose/H+ transporters in Plants. In vivo and in vitro mutagenesis of the lactose/H+ and melibiose/Na+ (H+) transporters of E. coli has identified individual amino acid residues alterations of which affect sugar and/or cation recognition and parameters of transport. Most of the bacterial transport proteins have been identified and the lactose/H+ transporter has been purified. The directions of future investigations are discussed.

Amino Acid Sequence↗
PubMed1981

Primordial transport of sugars and amino acids via Schiff bases.

Experimental support is given for a model concerning the origin of a primordial transport system. The model is based on the facilitated diffusion of amino acids stimulated by aliphatic aldehyde carriers and sugars stimulated by aliphatic amine carriers. The lipid-soluble diffusing species is the Schiff base. The possible role of this simple transport system in the origin of an early protocell is discussed.

Amino Acids↗
PubMed1989

Inhibition of adenosine deaminase activity reveals an intense active transport of adenosine into neurons in primary cultures.

It is often assumed that adenosine transport into brain cells occurs by facilitated diffusion and that the continued net uptake of adenosine depends on its subsequent metabolism, which keeps the intracellular concentration of unmetabolized adenosine low and thus maintains a concentration gradient. If that is the case, inhibition of adenosine metabolism should decrease uptake. We have previously reported a considerable deamination of accumulated adenosine to inosine in primary cultures of cerebral cortical neurons. A relatively specific adenosine deaminase inhibitor, 2'-deoxycoformycin, was used in the present study. In the presence of this drug, the adenosine content (pool size) increased many fold without any decrease in total influx of adenosine. Influx of accumulated adenosine took place against a concentration gradient, demonstrating that a metabolic degradation of accumulated adenosine is not required to drive adenosine uptake. This does not preclude that under normal conditions some adenosine may get into the cells by diffusion.

Adenosine↗
PubMed1989

Manganese(II) dynamics and distribution in glial cells cultured from chick cerebral cortex.

The kinetics of manganese(II) ion uptake and efflux have been investigated using tracer 54Mn(II) with glial cells cultured from chick cerebral cortex in chemically defined medium. The initial velocity of Mn(II) uptake versus [Mn(II)] exhibit saturation, with an apparent S0.5 approximately 18(+/- 3) microM. Both the rate and extent of Mn(II) uptake are inhibited by Ca(II), either added externally or preloaded into the glial cells. Preloading of glia with Mn(II) also inhibits the rate of external 54Mn(II) uptake. Zn(II) inhibits but Cu(II) activates Mn(II) uptake. Efflux of Mn(II) from preloaded cells occurs as a biphasic process, with rapid release of 30-40% of total cell Mn(II), then much slower release of the remainder. Permeabilization of cells with dextran sulfate also rapidly released ca. 30% of total cell Mn(II). High external Mn(II) enhanced both the rate and extent of Mn(II) efflux. CCCP, an uncoupler of oxidative phosphorylation, inhibited both Mn(II) uptake and efflux significantly, but addition of cyanide, ouabain, insulin, hydrocortisone, K+, or Nd(III) had no effect on either process. Taken together, these data suggest a model in which Mn(II) is brought across the plasma membrane by facilitated diffusion, binds to cytosolic protein sites, and is partitioned into the mitochondria by an active transport mechanism. The fact that the Mn(II) flux rates observed with cultured glia are much faster than those reported for overall uptake and efflux of brain Mn(II) in vivo suggests that the blood-brain barrier may play a significant role in determining these latter rates in whole animals.

Adenosine Triphosphate↗
PubMed1990

Cu(II) and Zn(II) ions alter the dynamics and distribution of Mn(II) in cultured chick glial cells.

Previous studies revealed that Mn(II) is accumulated in cultured glial cells to concentrations far above those present in whole brain or in culture medium. The data indicated that Mn(II) moves across the plasma membrane into the cytoplasm by facilitated diffusion or counter-ion transport with Ca(II), then into mitochondria by active transport. The fact that 1-10 microM Mn(II) ions activate brain glutamine synthetase makes important the regulation of Mn(II) transport in the CNS. Since Cu(II) and Zn(II) caused significant changes in the accumulation of Mn(II) by glia, the mechanisms by which these ions alter the uptake and efflux of Mn(II) ions has been investigated systematically under chemically defined conditions. The kinetics of [54MN]-Mn(II) uptake and efflux were determined and compared under four different sets of conditions: no adducts, Cu(II) or Zn(II) added externally, and with cells preloaded with Cu(II) or Zn(II) in the presence and absence of external added metal ions. Zn(II) ions inhibit the initial velocity of Mn(II) uptake, increase total Mn(II) accumulated, but do not alter the rate or extent Mn(II) efflux. Cu(II) ions increase both the initial velocity and the net Mn(II) accumulated by glia, with little effect on rate or extent of Mn(II) efflux. These results predict that increases in Cu(II) or Zn(II) levels may also increase the steady-state levels of Mn(II) in the cytoplasmic fraction of glial cells, which may in turn alter the activity of Mn(II)-sensitive enzymes in this cell compartment.

Animals↗
PubMed1987

Uptake of L-threo-dihydroxyphenylserine into human brain synaptosomes.

Out of four diastereoisomers of 3,4-dihydroxyphenylserine (DOPS), L-threo- and L-erythro-isomer were found to be taken up into human brain synaptosomes. The uptake of L-threo-DOPS was dependent on the temperature and sensitive to the metabolic inhibitors. The L-threo-DOPS uptake proved to be saturable and carrier-mediated transport with two different kinetic characteristics; a high-affinity and low-capacity and a low-affinity and high-capacity system. The apparent Km values of these two systems were obtained to be 28.6 microM and 2.47 mM, respectively. The high-affinity transport was inhibited by glycine, L-tyrosine, L-proline, L-serine, L-Dopa, L-tryptophan, and L-phenylalanine. The inhibition by L-tyrosine was competitive in regard to L-threo-DOPS. The L-threo-DOPS uptake was inhibited by 2,4-dinitrophenol, sodium cyanide and other uncouplers of oxidative phosphorylation and by ouabain, an inhibitor of Na+, K+-ATPase, indicating that the uptake is coupled to ATP hydrolysis. On the other hand, L-threo-DOPS uptake by the low-affinity system was not inhibited by metabolic inhibitors, indicating that it may be facilitated diffusion common to high concentrations of L-amino acids.

Amino Acids↗
PubMed1995

Ion efflux systems involved in bacterial metal resistances.

Studying metal ion resistance gives us important insights into environmental processes and provides an understanding of basic living processes. This review concentrates on bacterial efflux systems for inorganic metal cations and anions, which have generally been found as resistance systems from bacteria isolated from metal-polluted environments. The protein products of the genes involved are sometimes prototypes of new families of proteins or of important new branches of known families. Sometimes, a group of related proteins (and presumedly the underlying physiological function) has still to be defined. For example, the efflux of the inorganic metal anion arsenite is mediated by a membrane protein which functions alone in Gram-positive bacteria, but which requires an additional ATPase subunit in some Gram-negative bacteria. Resistance to Cd2+ and Zn2+ in Gram-positive bacteria is the result of a P-type efflux ATPase which is related to the copper transport P-type ATPases of bacteria and humans (defective in the human hereditary diseases Menkes' syndrome and Wilson's disease). In contrast, resistance to Zn2+, Ni2+, Co2+ and Cd2+ in Gram-negative bacteria is based on the action of proton-cation antiporters, members of a newly-recognized protein family that has been implicated in diverse functions such as metal resistance/nodulation of legumes/cell division (therefore, the family is called RND). Another new protein family, named CDF for 'cation diffusion facilitator' has as prototype the protein CzcD, which is a regulatory component of a cobalt-zinc-cadmium resistance determinant in the Gram-negative bacterium Alcaligenes eutrophus. A family for the ChrA chromate resistance system in Gram-negative bacteria has still to be defined.

ATP-Binding Cassette Transporters↗
PubMed1995

Alanine transport across the human placental brush border membrane and the role of SH groups in carrier function.

We have determined the kinetic characteristics of alanine transport into brush border membrane vesicles (BBMV) of human full term placenta and identified functional groups of the carrier proteins that are important for transport function. Alanine influx into BBMV was found to be mediated by two transport systems with different kinetic features and distinct substrate specificities. An uphill operating electrogenic Na(+)-dependent cotransport system could be kinetically separated from a Na(+)-independent facilitated diffusion system. The Na(+)-dependent transporter mediates Na(+)-alanine cotransport with a 1:1 flux coupling ratio (Hill coefficient 1.13 +/- 0.12) and a Km for alanine of 0.45 +/- 0.06 mmol/l. Half-maximal stimulation of Na(+)-dependent alanine influx was observed at a Na+ concentration (NaCl) of 51.4 +/- 1.3 mmol/l. A variety of group specific reagents were used to identify functional groups in the transport proteins. Only compounds reacting with SH-residues (NEM, DTNB, PCMBS) or NH2-groups (PITC) were found to affect Na+ dependent and Na+ independent alanine transport. The EC50 value for inhibition of alanine influx by PCMBS was 450 +/- 48 mumol/l. Chemical modifications of SH-groups by PCMBS caused a significant reduction (p < 0.005) in the Vmax for Na(+)-dependent alanine influx from 0.57 +/- 0.06 to 0.16 +/- 0.05 nmol.mg protein-1.10s-1 without affecting significantly the Km value. Inhibition by PCMBS was reversed by treatment of BBMV with DTT. When the substrate binding site of the transporter was protected by alanine or leucine, PCMBS still, blocked transport function, indicating that the crucial SH groups are not located within the substrate binding site of the transport proteins.

4-Chloromercuribenzenesulfonate↗
PubMed1988

The blood-nerve barrier is rich in glucose transporter.

The glucose transporter of the facilitated diffusion type has been localized in sections of innervated rat diaphragm muscle and sciatic nerve by immunofluorescence, using affinity-purified antibodies against both the entire transporter and the carboxy-terminal peptide. In both tissues the transporter was very abundant in the perineurial sheath of cells surrounding the nerve fibres. The transporter also appeared to be abundant in the endoneurial blood vessels of the sciatic nerve. The identity of the antigen as the glucose transporter was established by extracting sciatic nerve with sodium dodecylsulphate and immunoblotting the extract. A single reactive polypeptide with the expected molecular weight of 55,000 was found. The high concentration of glucose transporter in the cells of the blood-nerve barrier presumably ensures an adequate supply of glucose to the nerve fibres.

Animals↗
PubMed1977

Models for the active transport of cations...the steady-state analysis.

We summarise the progress that has been made in the analysis of active transport models, at the steady-state level. The two general classes of such model, counter-and co-transport, can be treated by a kinetic analysis which makes no assumptions as to the symmetry or asymmetry of the systems nor as to the presence of any particular rate-limiting steps. Precisely the same formalism is obeyed for primary active transport as for secondary active transport. Both are merely a generalisation of facilitated diffusion, in that they follow directly from accepted properties of carrier models. How affinities of such carriers for their substrates affect the efficiency of active transport is discussed and it is shown that in a number of cases, the affinity changes that the carrier demonstrates arise from inherent properties of the free carrier and not from any "high energy" properties of the chemical reactants. Methods of obtaining the kinetic parameters of the system from experimental data are reviewed, together with methods for testing and characterising the different transport models.

Binding Sites↗
PubMed1976

The role of thiol groups in nucleoside transport.

(1) The inactivation of various forms of nucleoside transport with reagents blocking thiol groups was studied in whole cells of E. coli B. No positive correlation between the efficiency of active transport and the extent or rate of inactivation could be demonstrated. (2) The most efficient constitutive nucleoside-transporting system was found to comprise a specific thiol component characterized by low rate of inactivation with N-ethylmaleimide; the less efficient inducible transport and the facilitated diffusion of guanosine require the integrity of another thiol component which is rapidly inactivated with N-ethylmaleimide. (3) The constitutive nucleoside-transporting system is completely inactivated with T4 phage, while other modes of nucleoside transport are much less affected. (4) Inactivation of constitutive transporting system in cells exposed to N-ethylmaleimide for a limited period of time continues long after the inhibitor has been removed, indicating storage of the inhibitor in some cellular compartment. Addition of dithiothreitol stops the inactivation immediately.

Arsenates↗
PubMed1976

Regulation of the D-glucose transport system in isolated fat cells.

Recent technical advances have yielded considerable new biochemical insights into the hexose transport systems of both brown and white fat cells. In the present studies a novel filtration method was used to monitor initial rates of 3-O-(3H)methylglucose uptake in isolated white fat cells. Transport of 3-O-methylglucose, a non-metabolizable analogue of glucose, occurred by facilitated diffusion, was inhibited by glucose, phloridzin, cytochalasin B and dipyridamole, and was rapidly stimulated by insulin as well as lectins. Total 3-O-methylglucose uptake in white fat cells could be attributed to two kinetically distinct processes in addition to a certain degree of diffusion. Two important new features of glucose transport in fat cells have been discovered. First, in both brown and white fat cells transport per se does not appear to be necessarily rate-limiting for further glucose metabolism. Thus vitamin K5, which markedly increases glucose oxidation by brown fat cells, did not affect the glucose transport system activity. Glucose utilization can apparently be significantly enhanced in fat cells by agents which either increase transport system activity or intracellular enzyme activity. Second, the transport system itself, whether in the basal state or after activation by insulin, lectins, or oxidants, is resistant to sulfhydryl reagents such as N-ethylmaleimide, while the increase in transport activity due to these agents is exquisitely sensitive to sulfhydryl blockage. N-ethylmaleimide blocks the stimulatory effect of insulin on transport whereas addition of insulin to fat cells prior to the reagent completely protects against this inhibitory effect. Further, N-ethylmaleimide prevents the elevated rates of transport system activity due to insulin (or other agents) from returning to basal levels once the cells are washed free of hormone. These data are consistent with the concept that activation of the transport system involves oxidation of key membrane sulfhydryls to the disulfide form, but alternative models are also possible. In any case, these findings provide a possible biochemical clue for future studies designed to identify the specific component(s) involved in the regulatory mechanism which modulates transport of glucose in isolated fat cells.

Adipose Tissue↗
PubMed1984

Ouabain-resistant Na+, K+ transport system in mouse NIH 3T3 cells.

It is shown that the ouabain-resistant (OR) furosemide-sensitive K+(Rb+) transport system performs a net efflux of K+ in growing mouse 3T3 cells. This conclusion is based on the finding that under the same assay conditions the furosemide-sensitive K+(Rb+) efflux was found to be two- to threefold higher than the ouabain-resistant furosemide-sensitive K+(Rb+) influx. The ouabain-resistant furosemide-sensitive influxes of both 22Na and 86Rb appear to be Cl- dependent, and the data are consistent with coupled unidirectional furosemide-sensitive influxes of Na+, K+ and Cl- with a ratio of 1:1:2. However, the net efflux of K+ performed by this transport system cannot be coupled to a ouabain-resistant net efflux of Na+ since the unidirectional ouabain-resistant efflux of Na+ was found to be negligible under physiological conditions. This latter conclusion was based on the fact that practically all the Na+ efflux appears to be ouabain-sensitive and sufficient to balance the Na+ influx under such steady-state conditions. Therefore, it is suggested that the ouabain-resistant furosemide-sensitive transport system in growing cells performs a facilitated diffusion of K+ and Na+, driven by their respective concentration gradients: a net K+ efflux and a net Na+ influx.

Animals↗
PubMed1979

A mathematical model for membrane transport of amino acid and Na+ in vesicles.

A model with a carrier having sites for both amino acid and Na+ can account for AIB (alpha-aminoisobutyric acid) transport kinetics observed in membrane vesicles from SV3T3 (simian virus 40-tranformed Balb/c33 cells) and 3T3 (the parent cell line). The main feature of this cotransport model is that Na+ binding to carrier decreases the effective Km for AIB transport. Na+ transport kinetics observed in both vesicle systems can be described by passive (possibly facilitated) diffusion. The lag of Na+ transport across the membrane compared to that for AIB, coupled to the Na+-dependent decrease in the Km for AIB, accounts for the overshoot in intravesicular AIB observed for SV3T3 in the presence of an initial Na+ gradient. Extra-vesicular Na+ maintains a decrease in the Km for AIB influx before intra-vesicular Na+ has accumulated to balance it with a comparable decrease in the Km for AIB efflux. 3T3 vesicles display little overshoot, and this finding can be explained mostly by a lower carrier affinity for Na+.

Aminoisobutyric Acids↗
PubMed1977

Comparative study of the thiourea carrier in erythrocytes.

A densimeter technique was used to measure the rate of exit of thiourea from erythrocytes of various species of mammals. The cells were first equilibrated with a 200 mM thiourea solution in 1% NaCl. An aliquot of these cells was added to 1% NaCl containing 4.6-23.1 mM thiourea. Facilitated diffusion was demonstrated in each case. Using exit times or initial rates, calculations of half-saturation constants (phi) in mM and maximum transport rates (K) in isotones per min were made by three different methods. The following values were obtained: human-phi=60, 42, 35; K=1.2, 2.9, 0.9; rabbit-phi=46, 33, 32; K=0.8, 2.1, 0.8; mouse-phi=46, 40, 30; K=3.4, 8.5, 3.2; rat-phi=65, 42, 23; K=6.1, 15.3, 3.7; ox-phi=107, 63, 88; K=0.6, 1.4, 0.4; sheep-phi=56, 38, 56; K=0.9, 2.2, 0.6; and pig-phi=110, 64, 49; K=1.6, 3.6, 1.1.

Animals↗
PubMed1975

A Na+-independent, phloretin-sensitive monosaccharide transport system in isolated intestinal epithelial cells.

A monosaccharide transport system in addition to the active Na+-dependent system characteristic of the brush border surface of vertebrate intestinal tissue has been identified in isolated chick intestinal epithelial cells. The newly described system differs in several characteristics from the Na+-dependent process, including function in the absence of Na+; a high sensitivity to phloretin, relative insensitivity to phlorizin; different substrate specificity; and a very high KT and Vmax. The system apparently functions only in a facilitated diffusion manner so that it serves to move monosaccharide across the cell membrane down its chemical gradient. An appreciable fraction of total sugar efflux occurs via the Na+-independent carrier from cells which have accumulated sugar to a steady state. Phloretin selectively blocks this efflux so that a normal steady-state sugar gradient of seven- to eightfold is transformed to a new steady-state gradient which is greater than 14-fold. Locus of the new system is tentatively ascribed to the serosal cell surface where it would serve for monosaccharide transfer between enterocyte and lamina propria of the villus.

Animals↗
PubMed1975

Membrane proteins and urea and acetamide transport in the human erythrocyte.

Previous studies have shown that urea and acetamide traverse the erythrocyte membrane by way of facilitated diffusion. The nature of this selective pathway is unknown. The present studies investigate the effects of proteolytic enzymes and crosslinking agents on amide transport. Cleavage of the erythrocyte membrane surface by pronase or trypsin had no effect on urea and acetamide permeability or inhibition by phloretin. These findings suggest that the sialoglycopeptide segment of the sialoglycoproteins is not critical to urea and acetamide transport. In addition, extensive crosslinking of membrane proteins with glutaraldehyde had no effect on amide transport in the absence or presence of phloretin.

Acetamides↗
PubMed1986

Nucleoside transport in rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine and p-chloromercuriphenyl sulfonate.

The sensitivity of nucleoside transport by rat erythrocytes to inhibition by nitrobenzylthioinosine (NBMPR) and the slowly permeating organomercurial, p-chloromercuriphenyl sulfonate (pCMBS), was investigated. The dose response curve for the inhibition of uridine transport (100 microM) by NBMPR was biphasic--35% of the transport activity was inhibited with an IC50 value of 0.25 nM, but 65% of the activity remained insensitive to concentrations as high as 1 microM. These two components of uridine transport are defined as NBMPR-sensitive and NBMPR-insensitive, respectively. Uridine influx by both components was saturable and conformed to simple Michaelis-Menten kinetics, and was inhibited by other nucleosides. The uridine affinity of the NBMPR-sensitive transport component was threefold higher than for the NBMPR-insensitive transport mechanism (apparent Km for uridine 50 +/- 18 and 163 +/- 28 microM, respectively). The two transport systems also differed in their sensitivity to pCMBS. NBMPR-insensitive uridine transport was inhibited by pCMBS with an IC50 of approximately 25 microM, while 1 mM pCMBS had little effect on NBMPR-sensitive transport by intact cells. pCMBS inhibition was reduced in the presence of uridine and adenosine and reversed by the addition by beta-mercaptoethanol, suggesting that the pCMBS-sensitive thiol group is located on the exterior surface of the erythrocyte membrane within the nucleoside binding site of the transport system. Inhibition of uridine transport by NBMPR was associated with high-affinity [3H]NBMPR binding to the cell membrane (apparent Kd 46 +/- 25 pM). Binding of inhibitor to these sites was competitively blocked by uridine and inhibited by adenosine, thymidine, dipyridamole, dilazep and nitrobenzylthioguanosine. Assuming that each NBMPR-sensitive transport site binds a single molecule of NBMPR, the calculated translocation capacity of each site is 25 +/- 6 molecules/site per sec at 22 degrees C. pCMBS had no effect on [3H]NBMPR binding to intact cells but markedly inhibited binding to disrupted membranes indicating that the NBMPR-sensitive nucleoside transporter probably has a thiol group located on the inner surface of the membrane. Exposure of rat erythrocyte membranes to UV light in the presence of [3H]NBMPR resulted in covalent radiolabeling of a membrane protein(s) (apparent Mr on SDS gel electropherograms of 62,000). Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine. We conclude that nucleoside transport by rat erythrocytes occurs by two facilitated-diffusion systems which differ in their sensitivity to inhibition by both NBMPR and pCMBS.

4-Chloromercuribenzenesulfonate↗