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1.
A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain β-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by β-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H+/sugar cotransport. Entry of is less dependent on the need for proton reextrusion, probably because the stoichiometry of H+/substrate cotransport is greater for lactose than for ONPG. 相似文献
2.
Paulette W. Royt 《生物化学与生物物理学报:生物膜》1982,687(2):226-230
The inducible, nonenergy-requiring glucose transport system of the yeast Kluyveromyces lactis is inactivated upon starving cells of glucose by (1) transferring logarithmic phase glucose-grown cells to synthetic medium containing a nonglycolytic carbon source, and (2) upon transition of logarithmic phase glucose-grown cells to stationary phase. The steady-state accumulation of nonmetabolizeable 6-deoxyglucose and the apparent of transport of 6-deoxyglucose is the same in stationary phase cells and in logarithmic phase cells. The rate of transport is lower in the nongrowing cells. Restoration of activity requires energy and protein synthesis as well as inducer. 相似文献
3.
2-Deoxyglucose and were used to assess endotoxin-induced changes in glucose transport in rat adipocytes. 6 h after Escherichia coli endotoxin injection insulin-stimulated 2-deoxyglucose uptake was significantly depressed (), phosphorylation of 2-deoxyglucose was seemingly unimpaired; basal 3-methylglucose entry was significantly increased, insulin-stimulated uptake was unaltered. Insulin significantly reduced in control and endotoxin-treated cells. Cytochalasin B-insensitive uptake of both 2-deoxyglucose and 3-methylglucose, a small fraction of total transport, increased significantly in endotoxic cells. Endotoxin reduced spermine- and insulin-stimulated 2-deoxyglucose uptake to a similar extent. Results are consistent with the hypotheses that (1) a site of endotoxin-induced insulin resistance is at the cell membrane level and may reflect a decrease in number or activity of effective carrier units, rather than alterations in affinity, (2) endotoxin does not compromise the hexokinase system, (3) the cell membrane-localized effect of endotoxin on hexose transport is not necessarily mediated by the insulin receptor and (4) the entry of 2-deoxyglucose and 3-methylglucose may involve two separate transport systems. 相似文献
4.
(1) Alkyl sugar inhibition of d-allose uptake into adipocytes has been used to explore the spatial requirements of the external sugar transport site in insulin-treated cells. α-methyl and β-methyl glucosides show low affinity indicating very little space around C-1. The high affinity of d-glucosamine (Ki = 9.05 ± 0.66 mM) is lost by N-acetylation. shows no detectable affinity, indicating that a bulky group at C-2 is not accepted. Similarly (Ki = 42.1 ± 7.5 mM) has lower affinity than (Ki = 5.14 ± 0.32 mM) indicating very little space around C-2 but much more around C-3. A reduction in affinity does occur if a propyl group is introduced into the C-3 position. The Ki for is 11.26 ± 2.12 mM. (Ki = 87.2 ± 17.9 mM) and (Ki = 78.07 ± 12.6 mM) show low affinity compared with d-galactose and d-glucose, indicating steric constraints around C-6. High affinity is restored in (Ki = 4.66 ± 0.23 mM) possibly indicating a hydrophobic binding site around C-6). (2) In insulin treated cells (Ki = 6.11 ± 0.5 mM) and maltose (Ki = 23.5 ± 2.1 mM) are well accommodated by the site but trehalose shows no detectable inhibition. These results indicate that the site requires a specific orientation of the sugar as it approaches the transporter from the external solution. C-1 faces the inside while C-4 faces the external solution. (3) To determine the spatial and hydrogen bonding requirements for basal cells 40 μM was used as the substrate. Poor hydrogen bonding analogues and analogues with sterically hindering alkyl groups showed similar Ki values to those determined for insulin-treated cells. These results indicate that insulin does not change the specificity of the adipocyte transport system. 相似文献
5.
(1) 4,6-O-Ethylidene-d-glucose is a good inhibitor of adipocyte sugar transport from the external surface. Using radioactively labelled 4,6-O-methylidene-d-glucose we have shown that this compound is not taken up into cells by the hexose transporter but through a route which is insulin insensitive, d-glucose insensitive, temperature sensitive and which is slightly inhibited by phloretin. When efflux of 3-O-methyl-d-glucose is studied with 4,6-O-methylidene-d-glucose only present inside the cells then no detectable inhibition is observed indicating that this compound is a good side-specific analogue with a high affinity for only the external site of the hexose transporter. (2) Radioactively labelled alkyl-β-d-glucosides have been prepared. These also penetrate the adipocyte membrane by an insulin and d-glucose insensitive route. The half-times for equilibration with methyl-, n-propyl-, and are 255, 9.45 and 3.3 min, respectively, indicating that the uptake rates are dependent upon the size of the alkyl group. (3) The glucosides show poor inhibition of 3-O-methyl-d-glucose transport when added to the external solution only. When cells are preincubated with and an increase in the amount of inhibition of 3-O-methyl-d-glucosez uptake is observed. This increase in inhibition correlates well with the glucoside uptake rates and indicates that availability of the glucosides at the internal surface of the transporter is required for inhibition. This has been confirmed by measuring 3-O-methyl-d-glucose exit in the presence of at the internal surface only. Thus, is a good side-specific analogue with high affinity only for the internal site of the hexose transporter. (4) inhibition of d-allose transport is fully reversible. If cells are washed after a preincubation with the analogue then the inhibition is lost. The inhibition of d-allose transport is reduced competitively by 3-O-methyl-d-glucose. (5) 6-O-Propyl-d-galactose has low affinity for both internal and external sites. 相似文献
6.
The kinetics of α-methyl-d-glucoside accumulation by rat kidney cortex slices under conditions of varying extracellular pH are compared with values obtained at pH 7.4. At pH below 7.4 there is a diminished initial uptake and reduced influx of the sugar which results in a decrease in the steady-state intracellular pool. This was associated with a decrease in the of the entry process without affecting the apparent of transport. At pH 8 there is no change in the rate of glucoside entry. The efflux of the glucoside, however, is impaired and the steady-state concentration gradient becomes greater than that observed at pH 7.4. 相似文献
7.
Hubert Felle 《生物化学与生物物理学报:生物膜》1984,772(3):307-312
The amino acid transport across the plasmalemma of Riccia fluitans rhizoid cells has been further characterized by means of current-voltage ) analysis. On the basis of two cyclic transport models which include six different carrier states, the question is raised, whether the electrochemical pH-gradient drives a negatively charged carrier or a positively charged alanine-proton-carrier complex across the membrane. analysis shows that (1) the typical characteristic of l-alanine transport follows a sigmoid curve, (2) maximal accumulation of l-alanine within the cytoplasm is reached after about 1 hour, (3) the electrically accessible cytoplasmic l-alanine concentration is limited to about 20 mM, and (4) the steady-state saturation current depends directly on external l-alanine concentration. It is concluded that (a) these results are consistent with the predictions of the models for a negatively charged carrier, and (b) that the rate-limiting step involves the translocation of the ternary complex. 相似文献
8.
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
9.
V A Fried 《Journal of molecular biology》1977,114(4):477-490
A new type of lactose permease mutant, called lacYf, does not actively transport the usual substrates; but it does facilitate the entry of β-galactosides into Escherichia coli K-12. The kinetics of facilitated entry, as assayed by hydrolysis of o-nitrophenyl-β-d-galactopyranoside by intact cells are identical to those observed with wild-type permease. However, the mutant permease activity is not affected by SH reagents or the substrate analog β-d-galactosyl-1-thio-β-d-galactopyranoside which strongly inhibit wild-type activity. Furthermore, the kinetics of formation of permease in the mutant following addition of inducer and the kinetics subsequent to removal of inducer differ strikingly from those observed in wild-type strains. The results are consistent with a block in the maturation of permease in the mutant resulting in the accumulation of a large amount of permease precursor. Studies of the heterogenotes provide evidence for a subunit structure for the lactose permease. 相似文献
10.
(1) The for 1.3 mM D-allose uptake and efflux in insulin-stimulated adipocytes is 1.7 ± 0.1 min. In the absence of insulin mediated uptake of D-allose is virtually eliminated and the uptake rate () is near that calculated for nonmediated transport. The kinetic parameters for D-allose zero-trans uptake in insulin-treated cells are Kztoi = 271.3 ± 34.2 mM, Vztoi = 1.15 ± 0.12 mM · s?1. (2) A kinetic analysis of the single-gate transporter (carrier) model interacting with two substrates (or substrate plus inhibitor) is presented. The analysis shows that the heteroexchange rates for two substrates interacting with the transporter are not unique and can be calculated from the kinetic parameters for each sugar acting alone with the transporter. This means that the equations for substrate analogue inhibition of the transport of a low affinity substrate such as D-allose can be simplified. It is shown that for the single gate transporter the Ki for a substrate analogue inhibitor should equal the equilibrium exchange Km for this analogue. (3) Analogues substituted at C-1 show a fused pyranose ring is accepted by the transporter. 1-Deoxy-D-glucose is transported but has low affinity for the transporter. High affinity can be restored by replacing a fluorine in the β-position at C-1. The Ki for ; the Ki for . Replacing the ring oxygen also results in a marked reduction in affinity. The Ki for . (4) A hydroxyl in the gluco configuration at C-2 is not required as 2-deoxy-d-galactose (Ki = 20.75 mM) has a slightly higher affinity than d-galactose (Ki = 24.49 mM). A hydroxyl in the manno configuration at C-2 interferes with transport as d-talose (Ki = 35.4 mM) has a lower affinity than d-galactose. (5) d-Allose (Km = 271.3 mM) and 3-deoxy-d-glucose (Ki = 40.31 mM) have low affinity but high affinity is restored by substituting a fluorine in the gluco configuration at C-3. The Ki for . (6) Analogues modified at C-4 and C-6 do not show large losses in affinity. However, 6-deoxy-d-glucose (Ki = 11.08 mM) has lower affinity than d-glucose and 6-deoxy-d-galactose Ki = 33.97 mM) has lower affinity than d-galactose. Fluorine substitution at C-6 of d-galactose restores high affinity. The Ki for . Removal of the C-5 hydroxymethyl group results in a large affinity loss. The Ki. The Ki for . (7) These results indicate that the important hydrogen bonding positions involved in sugar interaction with the insulin-stimulated adipocytes transporter are the ring oxygen, C-1 and C-3. There may be a weaker hydrogen bond to C-6. Sugar hydroxyls in non-gluco configurations may sterically hinder transport. 相似文献
11.
Fanja Kesbeke Peter J.M. van Haastert 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1985,847(1):33-39
Extracellular cAMP induces an intracellular accumulation of cAMP and cGMP levels in Dictyostelium discoideum. cAMP is detected by cell-surface receptors which are composed of a class of fast-dissociating sites () and a class of slow-dissociating sites (). Exposure of D. discoideum cells to 1 mM cAMP for 30 min induces a reduction of cAMP binding (down-regulation; Klein, C. and Juliani, M.H. (1977) Cell 10, 329–335). The number of fast-dissociating sites was reduced by 80–90% in down-regulated cells. These sites are composed of two forms with high and low affinity which interconvert during the binding reaction. In down-regulated cells this transition still occurred in the residual sites. The accumulation of cellular cAMP levels induced by a saturating stimulus decreased by 80–90%. The number of slow-dissociating sites was not significantly reduced in down-regulated cells, but their affinity decreased about 10-fold. The accumulation of cellular cGMP levels induced by a saturating stimulus was not decreased; however, about 20-fold higher cAMP concentrations were required to induce the same response. These results demonstrate that the cAMP transduction pathways to adenylate cyclase and guanylate cyclase are down-regulated differently. Furthermore, the results suggest that the fast-dissociating sites are involved in the activation of adenylate cyclase, while the slow-dissociating sites are coupled to guanylate cyclase. 相似文献
12.
Unidirectional flux of solutes into the intestinal mucosal cells is determined by the rate of movement of these molecules across both an unstirred water layer and the microvillus membrane of the epithelial cell. Therefore, an equation is derived in this paper that describes the velocity of active transport as a function of the characteristics of both the transport carrier in the membrane and the resistance of the overlying unstirred water layer. Using this equation a series of curves are presented that depict the effect on the kinetics of active transport of varying the thickness (d) or surface area (Sw) of the unstirred water layer, the free diffusion coefficient (D) of the solute, the distribution of active transport sites along the villus (), the maximal transport velocity (Jmd) and the true Michaelis constant (Km). These theoretical curves illustrate the serious limitations inherent in interpretation of previously published data dealing with active transport processes in the intestine. 相似文献
13.
(1) In isolated chloroplasts (class B) electron flow is controlled mainly by the intrathylakoid pH (pHin). A decrease in pHin due to the light-driven injection of protons inside the thylakoid leads to the retardation of electron flow between two photosystems. This effect can be abolished by uncouplers or under photophosphorylation conditions (addition of Mg2+-ADP with Pi); Mg2+-ATP does not influence the steady-state rate of electron flow, (2) The steady-state pH difference, ΔpH, across the thylakoid membrane was estimated from quantitative analysis of the rate of P-700+ reduction. In chloroplasts, without adding Mg2+-ADP, ΔpH increases from 1.6 to 3.2 as the external pH rises from 6 to 9.5. Under the photophosphorylation conditions, ΔpH decreases showing a minimum at the external pH 7.5 (). (3) The value of photosynthetic control, K, measured as the ratio of the steady-state rates of P-700+ reduction in the presence of Mg2+-ADP (with Pi) and without adding Mg2+-ADP is dependent on external pH variations, showing a maximum value of at pHout 7.5. This pH dependence coincides with that of the ADP-stimulated ΔpH decrease. (4) Experiments with spin labels provide evidence that the light-induced changes in the thylakoid membrane are sensitive to the addition of uncouplers and are affected only slightly by the addition of Mg2+-ADP and Pi. 相似文献
14.
G.D. Holman 《生物化学与生物物理学报:生物膜》1980,599(1):202-213
Glucose transport in human erythrocytes is characterized by a marked asymmetry in the and values for entry and for exit. In addition, they show a high and a high for equilibrium exchange but low values for infinite cis and for infinite trans exit and entry. An allosteric pore model has been proposed to account for these characteristics. In this model, substrate-induced conformational changes destabilize the interfaces between protein subunits (the pore gates).Pores doubly occupied from inside destabilize the transport gates and result in high and high transport parameters. This effect is less marked when pores are doubly occupied from outside and therefore transport asymmetry results. 相似文献
15.
A number of lectins has been purified by affinity chromatography on O-glycosyl polyacrylamide gels. The lectins isolated (and the particular sugar ligands used in the affinity carriers) are as follows: Anguilla anguilla, serum (α-L-fucosyl-), Vicia cracca, seeds; Phaseolus lunatus, seeds; Glycine soja, seeds; Dolichos biflorus, seeds; Maclura pomifera, seeds; Sarothamnus scoparius, seeds; Helix pomatia, ablumin glands; Clitocybe nebularis, fruiting bodies (all ); Ricinus communis, seeds (β-lactosyl-); Onomis spinosa, root; Fomes fomentarius, fruiting bodies; Marasmius oreades, fruiting bodies (all ), Canavalia ensiformis, seeds, (i.e., concanavalin A) ().Physicochemical properties of Glycine soja, Dolichos bifloras, Phaseolus lunatus, Helix pomatia and Ricinus communis lectins correponded well to properties of the preparations studied earlier by other workers. For the other purified lectins the essential physicochemical data (sedimentation coefficient, molecular weight, subunit composition, electrophoretic patterns, amino acid composition, carbohydrate content, isoelectric point) were established and their precipitating, hemagglutinating and mitogenic activities determined. 相似文献
16.
C.L. Woldringh N.B. Grover R.F. Rosenberger A. Zaritsky 《Journal of theoretical biology》1980,86(3):441-454
The dimensions of Escherichia coli (strain H266) in transition between two states of balanced growth, were determined from electron micrographs of fixed cells by sampling the culture at various times following nutritional shift-up from a doubling time of 72 min to one of 24 min. Mean cell length rises immediately and overshoots its final steady-state value, cell diameter increases monotonically; both approach their asymptotic levels only after several hours.The results are compared with the dimensions predicted by each of two models of cell growth and morphogenesis in rod-shaped bacteria. The first attributes cell elongation to circular zones that double in number at a particular time during the cell cycle and which act at rates proportional to the growth rate; the second is similar, except that it considers surface growth rather than length extension as the active process, length being determined passively. Two possibilities are examined, that the zonal growth rate adjusts immediately to the new growth conditions, and that it does so gradually.The experimental data appear consistent with the gradual response version of the surface growth model. 相似文献
17.
18.
Uri Nudel Jane Salmon Eitan Fibach Masaaki Terada Richard Rifkind Paul A. Marks Arthur Bank 《Cell》1977,12(2):463-469
The accumulation of α- and β-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific α- and β-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me2SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of α-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in β-globin mRNA sequences is not detected until 20–24 hr after culture. In cells exposed to hemin, both α- and β-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of is maintained during induction. In maximally induced cells, the mRNA ratios are approximately 1 in cells induced by Me2SO and HMBA, and 0.66 and 0.3–0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of α- and β-like genes. In addition, the relative accumulation of α- and β-globin mRNAs in induced cells differs with various types of inducers. 相似文献
19.
John I. Mc Cormick Sandra S. Susten James H. Freisheim 《Archives of biochemistry and biophysics》1981,212(1):311-318
L1210/R81 lymphoma cells are resistant to methotrexate (MTX) by virtue of a 35-fold elevation in dihydrofolate reductase and an inability to transport the folate antagonist drug effectively. In a phosphate-containing buffer there was little or no influx into the resistant cells at either 1 or 50 μm MTX. Replacement of this buffer with a 4-(2-hydroxyethyl)-1-piperazine-N′-2-ethanesulfonic acid-Mg2+ system resulted in an apparent influx of MTX into the resistant cells. Under these conditions, L1210/R81 cells achieved an apparent steady state at an extracellular MTX concentration of 50 μm. The apparent steady-state level of 5 nmol cells was well below the intracellular level of dihydrofolate reductase (45 nmol/109 cells). Efflux experiments at the apparent steady state indicated that 60% of the MTX was very rapidly removed from the cells by washing. Over the range of the experiment a further 20% of the MTX effluxed more slowly (). The apparent influx into the resistant cells at 5 μm MTX was inhibited 13% by sodium azide (100 μm) and initially stimulated, then inhibited, by p-chloromercuriphenylsulfonic acid (100 μm). 5-Methyltetrahydrofolate (100 μm) had little effect on the process while aminopterin (100 μm) was inhibitory (68%). Kt and V values of 2 × 10?5m and 0.31 nmol cells/min, respectively, were determined for the apparent influx in cells. Comparison of apparent MTX influx in the resistant cells with MTX transport in the sensitive cells indicates profound differences in the two processes. The evidence suggests that the apparent influx in the former cell line may consist of MTX binding to the cell membrane together with a small degree of MTX influx into the intracellular compartment. 相似文献
20.
Peter J.M. Van Haastert 《Biochemical and biophysical research communications》1984,124(2):597-604
D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (Kd = 15 nM, ). A second class is fast dissociating ( about 1 s) and is composed of high affinity binding sites H (Kd ≈ 60 nM), and low affinity binding sites L (Kd = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors. 相似文献