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1.
A Carruthers 《Biochemistry》1991,30(16):3898-3906
Two classes of theoretical mechanisms for protein-mediated, passive, transmembrane substrate transport (facilitated diffusion) are compared. The simple carrier describes a carrier protein that exposes substrate influx and efflux sites alternately but never both sites simultaneously. Two-site models for substrate transport describe carrier proteins containing influx and efflux sites simultaneously. Velocity equations describing transport by these mechanisms are derived. These equations take the same general form, being characterized by five experimental constants. Simple carrier-mediated transport is restricted to hyperbolic kinetics under all conditions. Two-site carrier-mediated transport may deviate from hyperbolic kinetics only under equilibrium exchange conditions. When both simple- and two-site carriers display hyperbolic kinetics under equilibrium exchange conditions, these models are indistinguishable by using steady-state transport data alone. Seven sugar transport systems are analyzed. Five of these systems are consistent with both models for sugar transport. Uridine, leucine, and cAMP transport by human red cells are consistent with both simple- and two-site models for transport. Human erythrocyte sugar transport can be modeled by simple- and two-site carrier mechanisms, allowing for compartmentalization of intracellular sugars. In this instance, resolution of the intrinsic properties of the human red cell sugar carrier at 20 degrees C requires the use of submillisecond transport measurements. 相似文献
2.
The kinetic behavior of five models for biological transport, only one of which is based on the classical carrier mechanism, is investigated. All give hyperbolic substrate saturation curves in accord with experimental observations on many systems. Several simple kinetic tests with substrates and competitive inhibitors serve to exclude or confirm proposed models. The tests involve measuring rates of efflux of radioactive substrate in the presence of (i) a competitive inhibitor outside the cell; (ii) inhibitor inside and outside; and (iii) unlabeled substrate outside. Rules for testing hypothetical mechanisms are presented in tables which may be consulted directly, disregarding the mathematical derivation. 相似文献
3.
This study examines inhibitions of human erythrocyte D-glucose uptake at ice temperature produced by maltose and cytochalasin B. Maltose inhibits sugar uptake by binding at or close to the sugar influx site. Maltose is thus a competitive inhibitor of sugar uptake. Cytochalasin B inhibits sugar transport by binding at or close to the sugar efflux site and thus acts as a noncompetitive inhibitor of sugar uptake. When maltose is present in the uptake medium, Ki(app) for cytochalasin B inhibition of sugar uptake increases in a hyperbolic manner with increasing maltose. When cytochalasin B is present in the uptake medium, Ki(app) for maltose inhibition of sugar uptake increases in a hyperbolic manner with increasing cytochalasin B. High concentrations of cytochalasin B do not reverse the competitive inhibition of D-glucose uptake by maltose. These data demonstrate that maltose and cytochalasin B binding sites coexist within the glucose transporter. These results are inconsistent with the simple, alternating conformer carrier model in which maltose and cytochalasin B binding sites correspond to sugar influx and sugar efflux sites, respectively. The data are also incompatible with a modified alternating conformer carrier model in which the cytochalasin B binding site overlaps with but does not correspond to the sugar efflux site. We show that a glucose transport mechanism in which sugar influx and sugar efflux sites exist simultaneously is consistent with these observations. 相似文献
4.
Mercedes Hernandez-Asensio Francisca F. Del Campo 《Archives of biochemistry and biophysics》1980,200(1):309-318
The rate of α-methylglucoside efflux from wild-type cells of Escherichia coli K-12 is enhanced by different substrates, as long as they are readily respired. A similar enhancement takes place in strains with impaired oxidative phosphorylation (unc mutants), regardless of their being able (strains AN120, N144, and AN382) or unable (strain NR70) to energize the membrane through respiratory electron flow. The uncouplers carbonylcyanide-m-chlorophenylhydrazone and tetrachlorosalicylanilide do not diminish the efflux acceleration in wild-type strains or unc mutants. However, the stimulation of α-methylglucoside efflux does not occur in the mutant AN59 which cannot perform a normal respiratory electron transport due to a defective synthesis of ubiquinone. The failure to stimulate the efflux is observed with succinate, which is a typical substrate of respiration, as well as with substrates which can yield ATP both at respiratory and substrate levels such as gluconate or glycerol. Moreover, potassium cyanide nullifies the acceleration of α-methylglucoside efflux caused in any type of strain and by any substrate. These results show that neither ATP nor an energized state of the membrane appears to be needed for respiration to accelerate α-methylglucoside release from E. coli cells, and question the existence of any energy-requiring reaction for αMG exit, previously proposed by other authors. 相似文献
5.
The effect of ethanol on the transport of 5-methyltetrahydrofolate in freshly isolated hepatocytes in vitro resulted in about a 30% increase in accumulation of substrate. It was shown that this was not due to differences in metabolism, nor to an inhibition of efflux. Preincubation with 40 mm ethanol for 45 min resulted in a significantly increased rate of entry of 5-methyltetrahydrofolate into the cells. The stimulatory effect was specific to 5-methyltetrahydrofolate since ethanol inhibited uptake of folate and methotrexate. The increased uptake was due to metabolism of ethanol as shown by studies with pyrazole. Also, the n-alkanols, propanol through pentanol, and sorbitol but not methanol were stimulatory. Anaerobiosis and sodium azide stimulated uptake of 5-methyl-tetrahydrofolate but were inhibitory to methotrexate uptake. These data, taken together, suggest that the ethanol effect is due to increased entry of 5-CH3-H4PteGlu into the cells possibly as the result of an increased cellular ratio. 相似文献
6.
《Bioorganic & medicinal chemistry》2016,24(24):6379-6389
A major obstacle confronting the discovery and development of new antibacterial agents to combat resistant Gram-negative (GN) organisms is the lack of a rational process for endowing compounds with properties that allow (or promote) entry into the bacterial cytoplasm. The major permeability difference between GN and Gram-positive (GP) bacteria is the GN outer membrane (OM) which is a permeability barrier itself and potentiates efflux pumps that expel compounds. Based on the fact that OM-permeable and efflux-deleted GNs are sensitive to many anti-GP drugs, recent efforts to approach the GN entry problem have focused on ways of avoiding efflux and transiting or compromising the OM, with the tacit assumption that this could allow entry of compounds into the GN cytoplasm. But bypassing the OM and efflux obstacles does not take into account the additional requirement of penetrating the cytoplasmic membrane (CM) whose sieving properties appear to be orthogonal to that of the OM. That is, tailoring compounds to transit the OM may well compromise their ability to enter the cytoplasm. Thus, a Gestalt approach to understanding the chemical requirements for GN entry seems a useful adjunct. This might consist of characterizing compounds which reach the cytoplasm, grouping (or binning) by routes of entry and formulating chemical ‘rules’ for those bins. This will require acquisition of data on large numbers of compounds, using non-activity-dependent methods of measuring accumulation in the cytoplasm. 相似文献
7.
Lyle W. Horn 《The Journal of membrane biology》1986,89(2):185-192
Summary It is shown that the axoplasmic composition of acidic and neutral amino acids can be controlled effectively by the method of internal dialysis. Direct assay for specific binding and measurement of diffusion coefficients in axoplasm show that there is no significant binding or compartmentalization of amino acids. The dependence of amino acid efflux on substrate concentration can be measured under well-defined, true steady-state conditions. The taurine efflux-concentration relation in theMyxicola giant axon conforms to a second-order Hill equation. This fact is consistent with either a cooperative process or a mechanism in which membrane translocation is not the rate-controlling step. The effluxes of taurine and glycine from squid axon are an order of magnitude smaller than inMyxicola. The efflux-concentration relations are essentially linear up to 200mm substrate concentration. This result may be produced by specific transporters which have very high asymmetry, or by simple diffusive leak in the absence of specific transporters. 相似文献
8.
Axoplasmic free magnesium levels and magnesium extrusion from squid giant axons 总被引:5,自引:4,他引:1
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《The Journal of general physiology》1976,68(2):159-178
The free magnesium concentration in the axoplasm of the giant axon of the squid, Loligo pealei, was estimated by exploting the known sensitivity of the sodium pump to intracellular Mg2+ levels. The Mg- citrate buffer which, when injected into the axon, resulted in no change in sodium efflux was in equilibrium with a Mg2+ level of about 3- -4 mM. Optimal [Mg2+] for the sodium pump is somewhat higher. Total magnesium content of axoplasm was 6.7 mmol/kg, and that of hemolymph was 44 mM. The rate coefficient for 28Mg efflux was about 2 X 10(-3) min-u for a 500-mum axon at 22-25degreesC, with a very high temperature coefficient (Q10=4-5). This efflux is inhibited 95% by injection of apyrase and 75% by removal of external sodium, and seems unaffected by membrane potential or potassium ions. Increased intracellular ADP levels do not affect Mg efflux nor its requirement for Na+/o, but extracellularl magnesium ions do. Activation of 28Mg efflux by Na+/o follows hyperbolic kinetics, with Mg2+/o reducing the affinity of the system for Na+/o. Lanthanum and D600 reversibly inhibit Mg efflux. In the absence of both Na+ and Mg2+, but not in their presence, removal of Ca2+ from the seawater vastly increased 28Mg efflux; this efflux was also strongly inhibited by lanthanum. A small (10(-14) mol cm-2) extra Mg efflux accompanies the conduction of an action potential. 相似文献
9.
Low levels of Cd2+ (1-5 microM) produce rapid swelling of mitochondria, which is respiration-dependent and uncoupler-sensitive. No cation requirement is apparent, since the swelling occurs in a medium containing only sucrose and the respiratory substrate. The swelling is inhibited by ruthenium red, suggesting that this effect of Cd2+ requires its entry into mitochondria. In medium containing 9 mM K+, addition of Cd2+ along with ruthenium red increases the rate of K+ influx threefold. In the presence of K+, Rb+ or Li+, but not of Na+, addition of Cd2+ produces first efflux of H+ into the medium followed by discharge of the pH gradient or uncoupling. Only the latter effect is inhibited by ruthenium red, showing that the efflux and influx of H+ are independent reactions. The H+ efflux appears to be an antiport response to the induced K+ entry. Its activation by Cd2+ is similar to the known effect of p-chloromercuriphenyl sulfonate. The H+ influx or uncoupling appears to result from binding of Cd2+ to some matrix-facing membrane site, perhaps the dithiol group on coupling factor B, and may relate to apparent permeability changes associated Cd2+-induced swelling. 相似文献
10.
The strictly aerobic yeastCandida parapsilosis transports the nonmetabolizable monosaccharide 6-deoxy-D-glucose by an active process (inhibition by 2.4-dinitrophenol and
other uncouplers but not by iodoacetamide), the accumulation ratio decreasing with increasing substrate concentration. Measured
accumulation ratios are in agreement with those predicted from kinetic constants for influx and efflux. Energy for transport
is probably required in the translocation step. The maximum rate is temperature-dependent with a transition point at 21 °C.
the accumulation ratio is not, The uptake is most active at pH 4.5–8.5. It appears not to involve stoichiometric proton symport.
The transport system is shared by D-glucose, D-mannose, D-galactose and possibly maltose but not by fructose, sucrose or pentoses.
The apparent half-life of the transport system was 3.5–4 h. 相似文献
11.
ATP hydrolysis catalysed by the H+-ATPase of intact mitochondria can be induced by addition of ATP in the presence of valinomycin and KCl. This leads to an increase in intramitochondrial Pi and therefore allows investigation of potential Pi efflux pathways in intact mitochondria. Combining this approach with the direct measurement of both internal and external Pi, we have attempted to determine whether Pi efflux occurs via an atractyloside-sensitive transporter, by the classical operation of the Pi/H+ and Pi/dicarboxylate carriers, and/or by other mechanisms. Initial experiments re-examined the evidence that led to the current view that one efflux pathway for Pi is an atractyloside-sensitive ATP/ADP,0.5Pi transporter. No evidence was found in support of this efflux pathway. Rather, atractyloside-sensitivity of the low rate of Pi efflux observed in previous studies (oligomycin present) was accounted for by ATP entry on the well known ATP/ADP transport system followed by hydrolysis of ATP and subsequent Pi efflux. Thus, under these conditions, where ATP hydrolysis is not completely inhibited, Pi efflux becomes atractyloside sensitive most likely because this inhibitor blocks ATP entry, not because it directly inhibits Pi efflux. Substantial efflux of Pi from rat liver mitochondria is observed on generation of high levels of matrix Pi by ATP hydrolysis induced by valinomycin and K+ (oligomycin absent). A portion of this efflux can be inhibited by thiol-specific reagents at concentrations that normally inhibit the Pi/H+ and Pi/dicarboxylate carriers. However, a significant fraction of efflux continues even in the presence of p-chloromercuribenzoate, N-ethylmaleimide plus n-butylmalonate or mersalyl. The mersalyl-insensitive Pi efflux, which is also insensitive to carboxyatractyloside, is a saturable process, thus suggesting carrier mediation. During this efflux the mitochondrial inner membrane retains considerable impermeability to other low-molecular-weight anions (i.e., malate, 2-oxoglutarate). In conclusion, results presented here rule out an atractyloside-sensitive ATP/ADP,0.5Pi transport system as a mechanism for Pi efflux in rat liver mitochondria. Rather Pi efflux appears to occur on the classical Pi/H+ transport system as well as via a mersalyl-insensitive saturable process. The inhibitor-insensitive Pi efflux may occur on a portion of the Pi/H+ carrier molecules that exist in a state different from that normally catalysing Pi influx. Alternatively, a separate Pi efflux carrier may exist. 相似文献
12.
R M Johnstone 《Biochimica et biophysica acta》1975,413(2):252-264
Gramicidin induces a marked Na+-dependent efflux of amino acids from Ehrlich cells. In absence of Na+, gramicidin does not alter the efflux. In presence gramicidin, glycine efflux is inhibited by methionine and less so by leucine. Glycine efflux caused by HgCl2 is neither Na+ dependent nor inhibitable by amino acids. Neither efflux of inositol which is transported by an Na+-dependent route, nor efflux of several other solutes which are transported by Na+-independent routes, is affected by gramicidin. The antibiotic appears to permit a reversal in the direction of of the operation of the Na+-dependent amino acid transport system. The increased efflux is partly, but not entirely, due to an increase in the cellular Na+ concentration and a reduction of the electrochemical potential difference for Na+. 相似文献
13.
J A Barnett 《Journal of general microbiology》1975,90(1):1-12
The utilization of D-ribose by yeasts of the genus Pichia was examined with respect to aerobic growth, respiration and entry of ribose into the cells. Pichia etchellsii (CBS2011) could respire D-ribose, but not use it for aerobic growth. Pichia fermentans (CBS187) neither respired nor grew on D-ribose, though it entered the cells of this yeast either by simple diffusion, or possibly, by the D-glucose carrier, this having a very low affinity for D-ribose. Pichia pinus (CBS5097) respired and grew on D-ribose; kinetic evidence is given for this yeast having two ribose carriers, one inducible and the other constitutive. 相似文献
14.
Of the total one-way chloride fluxes (approximately 1.4 meq/liter cell water X min) in steady state human polymorphonuclear leukocytes bathed in 148 mM Cl media, approximately 70% behaves as self-exchange mediated by a nonselective anion carrier that is not inhibited by stilbene disulfonates. Five properties of this carrier-mediated exchange were investigated: substrate saturation is seen with respect to 36Cl influx as a function of the external Cl concentration [for normal-Cl cells, the apparent Km(Cl) is approximately 22 mM when Cl replaces para-amino- hippurate (PAH) and approximately 5 mM when Cl replaces glucuronate], and with respect to 36Cl efflux as a function of the concentration of internal Cl replacing PAH [apparent Km(Cl) congruent to 35 mM for cells bathed in 148 mM Cl]; there is trans stimulation of 36Cl influx by internal Cl (replacing PAH) with an apparent Km(Cl) congruent to 35 mM, and of 36Cl efflux by external Cl with an apparent Km(Cl) congruent to 22 mM (Cl replacing PAH) or approximately 5 mM (Cl replacing glucuronate); there is substrate competition between Cl and PAH, but the carrier appears devoid of affinity for glucuronate; influxes and effluxes mediated by the carrier are subject to competitive inhibition by extracellular alpha-cyano-4-hydroxycinnamate (CHC), with an apparent Ki congruent to 9 mM in Cl medium or approximately 1 mM in PAH medium (transport of the inhibitor itself is very slow); and internal Cl and external Cl or PAH undergo 1:1 countertransport, which is CHC sensitive. A simple equilibrium-competition model is proposed that accounts for all the extracellular ligand interactions presented for normal-Cl cells. Least-squares values of the carrier's true Michaelis constants for extracellular Cl, PAH, and CHC are 5.03 +/- 0.83, 50.3 +/- 14.9, and 0.29 +/- 0.09 mM, respectively. 相似文献
15.
J B van de Mortel D Mulders H Korthout A P Theuvenet G W Borst-Pauwels 《Biochimica et biophysica acta》1988,936(3):421-428
At pH 7, addition of glucose under anaerobic conditions to a suspension of the yeast Saccharomyces cerevisiae causes both a transient hyperpolarization and a transient net efflux of K+ from the cells. Hyperpolarization shows a peak at about 3 min and a net K+ efflux at 4-5 min. An additional transient hyperpolarization and net K+ efflux are found after 60-80 and 100 min, respectively. Addition of 2-deoxyglucose instead of glucose does not lead to hyperpolarization of the cells or K+ efflux. At low pH, neither transient hyperpolarization nor a transient K+ efflux are found. With ethanol as substrate and applying aerobic conditions, both a transient hyperpolarization and a transient K+ efflux are found at pH 7. The fluorescent probe 2-(dimethylaminostyryl)-1-ethylpyridinium appears to be useful for probing changes in the membrane potential of S. cerevisiae. It is hypothesized that the hyperpolarization of the cells is due to opening of K+ channels in the plasma membrane. Accordingly, the hyperpolarization of the cells at pH 7 is almost completely abolished by 1.25 mM K+, whereas the same amount of Na+ does not reduce the hyperpolarization. 相似文献
16.
Dose-response curves relating the external stimulus concentration to
receptor occupancy differ in two types of chemoreceptor organs. In
'concentration detectors' the receptor molecules at the receptor cell
membrane are directly exposed to the external stimulus concentration; these
organs exhibit the well-known hyperbolic dose-response relationship
reflecting the association-dissociation of stimulus and receptor molecules.
In contrast, 'flux detectors' accumulate the stimulus molecules in a
perireceptor compartment. In flux detectors, deactivation of stimulus
molecules may be in balance with arrival, as a prerequisite for producing a
constant effective stimulus concentration at constant adsorptive flux of
stimulus molecules. In a simple model of a flux detector in which receptor
molecules themselves catalyze the deactivation, the dose-response
relationship is linear. It reflects the rate of stimulus deactivation. If
the deactivation is catalyzed by a separate enzyme, the dose-response
relationship can be close to hyperbolic, or linear. In all cases, the
receptor molecules are maximally occupied if the adsorptive flux equals or
exceeds the maximum rate of stimulus deactivation. The time course of the
receptor potential recorded from moths' pheromone receptors depends on the
odor compound, which suggests that a peripheral process, possibly the
stimulus deactivation, is the slowest, rate-limiting process of the
transduction cascade. Further evidence comes from experiments with stimuli
oversaturating the mechanism responsible for the decline of the receptor
potential.
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17.
Control of Growth Rate by Initial Substrate Concentration at Values Below Maximum Rate 总被引:1,自引:1,他引:1
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The hyperbolic relationship between specific growth rate, mu, and substrate concentration, proposed by Monod and used since as the basis for the theory of steady-state growth in continuous-flow systems, was tested experimentally in batch cultures. Use of a Flavobacterium sp. exhibiting a high saturation constant for growth in glucose minimal medium allowed direct measurement of growth rate and substrate concentration throughout the growth cycle in medium containing a rate-limiting initial concentration of glucose. Specific growth rates were also measured for a wide range of initial glucose concentrations. A plot of specific growth rate versus initial substrate concentration was found to fit the hyperbolic equation. However, the instantaneous relationship between specific growth rate and substrate concentration during growth, which is stated by the equation, was not observed. Well defined exponential growth phases were developed at initial substrate concentrations below that required for support of the maximum exponential growth rate and a constant doubling time was maintained until 50% of the substrate had been used. It is suggested that the external substrate concentration initially present "sets" the specific growth rate by establishing a steady-state internal concentration of substrate, possibly through control of the number of permeation sites. 相似文献
18.
A key attribute of riverine food webs is the downstream movement of invertebrates via the water column, or invertebrate drift. Causes of drift include benthic predation, food limitation, and perhaps passive entry, which may occur when invertebrates lose their purchase on stream substrate. However, the relative importance of drift causes is unknown, as is whether the relative importance of drift causes varies across space. Combining observational data on invertebrate herbivore and predator guild densities with in‐stream experiments, we evaluated the relative importance of benthic predation, food limitation, and passive entry as proximate causes of drift for the herbivore guild across the canopy gradient of a montane stream. We found that 1) benthic predation and food limitation were both more important as causes of herbivore drift than passive entry; 2) drift caused by food limitation did not vary with riparian canopy, whereas herbivore density decreased with increasing riparian canopy, and 3) per capita drift increased linearly with increasing density, while per capita drift decreased in a negative hyperbolic fashion with increasing food, indicating that herbivore drift is proportional to herbivore density, and inversely proportional to food. We conclude that invertebrate herbivore drift was overwhelmingly an active process to improve fitness, and that herbivore food did not vary across the canopy gradient, likely because increased herbivory from larger herbivore populations at sunnier sites prevented food from accumulating. 相似文献
19.
L E Cornett J S Norris 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1987,186(2):157-164
In this study the mechanisms involved in alpha 1-adrenergic receptor-mediated Ca2+ mobilization at the level of the plasma membrane were investigated. Stimulation of 45Ca2+ efflux from saponin-permeabilized DDT1 MF-2 cells was observed with the addition of either the alpha 1-adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of [32P]NAD, pertussis toxin was found to catalyze ADP-ribosylation of a Mr = 40,500 (n = 8) peptide in membranes prepared from DDT1 MF-2 cells, possibly the alpha-subunit of Ni. However, stimulation of unidirectional 45Ca2+ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the alpha 1-adrenergic receptor to Ca2+ mobilization in DDT1 MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of the guanine nucleotide binding protein family. 相似文献
20.
Favari E Zimetti F Bortnick AE Adorni MP Zanotti I Canavesi M Bernini F 《FEBS letters》2005,579(29):6537-6542
We investigated the interaction of oxidized low density lipoprotein (OxLDL) with the ATP-binding cassette A1 (ABCA1) pathway in J774 macrophages. Cellular efflux to apolipoprotein AI (apo-AI) of OxLDL-derived cholesterol was lower than efflux of cholesterol derived from acetylated low density lipoprotein (AcLDL). ABCA1 upregulation by 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (cpt-cAMP) or 22 (R)-hydroxycholesterol (22-OH) and 9-cis retinoic acid (9cRA) increased the efflux to apo-AI of cellular sterols derived from AcLDL, but not of those from OxLDL. AcLDL, but not OxLDL, induced ABCA1 protein content and activity in J774. However, OxLDL did not influence J774 ABCA1 upregulation by cpt-cAMP or 22-OH/9cRA. We conclude that sterols released to cells by OxLDL are available neither as substrate nor as modulator of ABCA1. 相似文献