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1.
The phosphotransfer protein IIAGlc of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system plays a key role in the regulation of carbohydrate metabolism. Melibiose permease (MelB) is one among several permeases subject to IIAGlc regulation. The regulatory mechanisms are poorly understood; in addition, thermodynamic features of IIAGlc binding to other proteins are also unknown. Applying isothermal titration calorimetry and amine-specific cross-linking, we show that IIAGlc directly binds to MelB of Salmonella typhimurium (MelBSt) and Escherichia coli MelB (MelBEc) at a stoichiometry of unity in the absence or presence of melibiose. The dissociation constant values are 3–10 μm for MelBSt and 25 μm for MelBEc. All of the binding is solely driven by favorable enthalpy forces. IIAGlc binding to MelBSt in the absence or presence of melibiose yields a large negative heat capacity change; in addition, the conformational entropy is constrained upon the binding. We further found that the IIAGlc-bound MelBSt exhibits a decreased binding affinity for melibiose or nitrophenyl-α-galactoside. It is believed that sugar binding to the permease is involved in an induced fit mechanism, and the transport process requires conformational cycling between different states. Thus, the thermodynamic data are consistent with the interpretation that IIAGlc inhibits the induced fit process and restricts the conformational dynamics of MelBSt.  相似文献   

2.
A new oriented method using a diazonium salt reaction was developed for linking β 2-adrenoceptor (β 2-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β 2-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β 2-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×103/M for ephedrine and (3.80±0.02) ×103/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10−4 M. Thermodynamic studies showed that the binding of the two compounds to β 2-AR was a spontaneous reaction with exothermal processes. The ΔGθ, ΔHθ and ΔSθ for the interaction between ephedrine and β 2-AR were −(22.33±0.04) kJ/mol, −(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were −(21.17±0.02) kJ/mol, −(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β 2-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.  相似文献   

3.
Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane–based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine KM, Vmax, KD, and kobs values for different sugar substrates. KD and kobs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H+ symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H+ and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s−1) and for sugar translocation (2 s−1 − 30 s−1 for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.  相似文献   

4.
InEnterobacteriaceae the nonphosphorylated form of IIAG1c of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the Ma1K protein of the maltose transport system, respectively. In this report we show that the IIAGlc-Iike domain of the membrane-bound IIN-acetylglucosamine (IINag) of the PTS can replace IIAGlc in aSalmonella typhimurium crr mutant strain that lacks all soluble IIAGlc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose up take systems. TheStreptococcus thermophilus lactose transporter LacS, which also contains a IIAGlc-like domain, could not replace IIAGlc. Neither IINag nor LacS could replace IIAGlc in activation of adenylate cyclase.  相似文献   

5.
Characterization of the thermodynamics of DNA– drug interactions is a very useful part in rational drug design. Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and UV melting experiments have been used to analyze the multivalent (intercalation plus minor groove) binding of the antitumor antibiotic chartreusin to DNA. Using DNA UV melting studies in the presence of the ligand and the binding enthalpy determined by ITC, we determined that the binding constant for the interaction was 3.6 × 105 M–1 at 20°C, in a solution containing 18 mM Na+. The DNA–drug interaction was enthalpy driven, with a ΔHb of –7.07 kcal/mol at 20°C. Binding enthalpies were determined by ITC in the 20–35°C range and used to calculate a binding-induced change in heat capacity (ΔCp) of –391 cal/mol K. We have obtained a detailed thermodynamic profile for the interaction of this multivalent drug, which makes possible a dissection of ΔGobs into the component free energy terms. The hydrophobic transfer of the chartreusin chromophore from the solution to the DNA intercalating site is the main contributor to the free energy of binding.  相似文献   

6.
The binding of Ca2+ and Y3+ to an acidic glycoprotein from bovine cortical bone, bone sialoprotein, was determined from the titration curves at I 0·2 in the presence and absence of the cations. The binding of Y3+ was greater than that of Ca2+. The value for the association constant, k, for the interaction with Y3+ increased with pH, from log k 2·93 at pH3·4 to log k 3·50 at pH4·4, and the number of binding sites/mol. increased from 4·6 at pH3·4 to 9·1 at pH4·4. It is proposed that the binding site consists of three carboxyl groups, but it is likely that the binding is a strong electrostatic interaction rather than a co-ordination linkage. A chondroitin sulphate–protein complex also extracted from bovine cortical bone interacted with Y3+ and Ca2+ to a similar extent as did bone sialoprotein. It is suggested that these materials are present in bone at the resting and resorbing surfaces and that they contribute to the deposition of yttrium, americium and plutonium at these sites.  相似文献   

7.
A bioactive ingredient in an ethanol extract from the branch bark of cultivated mulberry Husang-32 (Morus multicaulis Perr.) was isolated using a macroporous resin column. The primary component, which was purified by semi-preparative high-performance liquid chromatography diode array detection (HPLC-DAD), was identified as mulberroside A (MA) by liquid chromatograph-mass spectrometer (LC-MS), 1H and 13C nuclear magnetic resonance (NMR) spectra. In total, 4.12 g MA was efficiently extracted from one kilogram of mulberry bark. The enzymatic analysis showed that MA inhibited the generation of dopachrome by affecting the activities of monophenolase and diphenolase of tyrosinase in vitro. This analysis indicated that MA and oxyresveratrol (OR), which is the the aglycone of mulberroside A, exhibited strong inhibition of the monophenolase activity with IC50 values of 1.29 µmol/L and 0.12 µmol/L, respectively. However, the former showed weaker inhibitory activity than the latter for diphenolase. For the monophenolase activity, the inhibitory activity of MA and OR was reversible and showed mixed type 1 inhibition. Additionally, the inhibition constant KI (the inhibition constant of the effectors on tyrosinase) values were 0.385 µmol/L and 0.926 µmol/L, respectively, and the KIS (the inhibition constants of the enzyme-substrate complex) values were 0.177 µmol/L and 0.662 µmol/L, respectively. However, MA showed competitive inhibition of diphenolase activity, and KI was 4.36 µmol/L. In contrast, OR showed noncompetitive inhibition and KI = KIS = 2.95 µmol/L. Taken together, these results provide important information concerning the inhibitory mechanism of MA on melanin synthesis, which is widely used in whitening cosmetics.  相似文献   

8.
Triapine® (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP)) is a drug in Phase II trials. One of its established cellular targets is the β2 subunit of ribonucleotide reductase that requires a diferric-tyrosyl-radical [(FeIII2-Y·)(FeIII2)] cofactor for de novo DNA biosynthesis. Several mechanisms for 3-AP inhibition of β2 have been proposed; one involves direct iron chelation from β2, whereas a second involves Y· destruction by reactive oxygen species formed in situ in the presence of O2 and reductant by Fe(II)-(3-AP). Inactivation of β2 can thus arise from cofactor destruction by loss of iron or Y·. In vitro kinetic data on the rates of 55Fe and Y· loss from [(55FeIII2-Y·)(55FeIII2)]-β2 under aerobic and anaerobic conditions reveal that Y· loss alone is sufficient for rapid β2 inactivation. OxyblotTM and mass spectrometric analyses of trypsin-digested inhibited β2, and lack of Y· loss from H2O2 and O2˙̄ treatment together preclude reactive oxygen species involvement in Y· loss. Three mammalian cell lines treated with 5 μm 3-AP reveal Y· loss and β2 inactivation within 30-min of 3-AP-exposure, analyzed by whole-cell EPR and lysate assays, respectively. Selective degradation of apo- over [(FeIII2-Y·)(FeIII2)]-β2 in lysates, similar iron-content in β2 immunoprecipitated from 3-AP-treated and untreated [55Fe]-prelabeled cells, and prolonged (12 h) stability of the inhibited β2 are most consistent with Y· loss being the predominant mode of inhibition, with β2 remaining iron-loaded and stable. A model consistent with in vitro and cell-based biochemical studies is presented in which Fe(II)-(3-AP), which can be cycled with reductant, directly reduces Y· of the [(FeIII2-Y·)(FeIII2)] cofactor of β2.  相似文献   

9.
In this report, we compared the effects on the growth of Lactobacillus plantarum of raising the medium molarity by high concentrations of KCl or NaCl and iso-osmotic concentrations of nonionic compounds. Analysis of cellular extracts for organic constituents by nuclear magnetic resonance spectroscopy showed that salt-stressed cells do not contain detectable amounts of organic osmolytes, whereas sugar-stressed cells contain sugar (and some sugar-derived) compounds. The cytoplasmic concentrations of lactose and sucrose in growing cells are always similar to the concentrations in the medium. By using the activity of the glycine betaine transport system as a measure of hyperosmotic conditions, we show that, in contrast to KCl and NaCl, high concentrations of sugars (lactose or sucrose) impose only a transient osmotic stress because external and internal sugars equilibrate after some time. Analysis of lactose (and sucrose) uptake also indicates that the corresponding transport systems are neither significantly induced nor activated directly by hyperosmotic conditions. The systems operate by facilitated diffusion and have very high apparent affinity constants for transport (>50 mM for lactose), which explains why low sugar concentrations do not protect against hyperosmotic conditions. We conclude that the more severe growth inhibition by salt stress than by equiosmolal concentrations of sugars reflects the inability of the cells to accumulate K+ (or Na+) to levels high enough to restore turgor as well as deleterious effects of the electrolytes intracellularly.  相似文献   

10.
A sugar-specific component of the lactose transport system in Staphylococcus aureus, Factor IIIlac, is phosphorylated as an intermediate in the over-all transfer of a phosphoryl group from PEP to lactose. P-IIIlac is isolated and shown to be a substrate for the final phosphoryl transfer reaction to sugar, catalyzed by Enzyme IIlac.  相似文献   

11.
The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a carboxyl-terminal regulatory domain (IIALacS) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIAGlc of Escherichia coli is the best-characterized member. On the basis of these similarities, it was anticipated that IIALacS would be able to perform one or more functions associated with IIAGlc, i.e., carry out phosphoryl transfer and/or affect other catabolic functions. The gene fragment encoding IIALacS was overexpressed in Escherichia coli, and the protein was purified in two steps by metal affinity and anion-exchange chromatography. IIALacS was unable to restore glucose uptake in a IIAGlc-deficient strain, which is consistent with a very low rate of phosphorylation of IIALacS by phosphorylated HPr (HPr~P) from E. coli. With HPr~P from S. thermophilus, the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIALacS (k1 = 4.3 × 102 M−1 s−1) and dephosphorylation of IIALacS~P by HPr (k−1 = 1.1 × 103 M−1 s−1) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIAGlc and HPr from E. coli. This finding suggests that IIALacS has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIAGlc, we constructed a double mutant [IIALacS(I548E/G556D)] that was predicted to have increased phosphoryl transfer activity. Indeed, the phosphorylation rate of IIALacS(I548E/G556D) by HPr~P increased (k1 = 4.0 × 103 M−1 s−1) and became nearly independent of the source of HPr~P (S. thermophilus, Bacillus subtilis, or E. coli). The increased phosphoryl transfer rate of IIALacS(I548E/G556D) was insufficient to complement IIAGlc in PTS-mediated glucose transport in E. coli. Both IIALacS and IIALacS(I548E/G556D) could replace IIAGlc, but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form.  相似文献   

12.
This work reports the biochemical and functional analysis of the Burkholderia cenocepacia J2315 bceN gene, encoding a protein with GDP-D-mannose 4,6-dehydratase enzyme activity (E.C.4.2.1.47). Data presented indicate that the protein is active when in the tetrameric form, catalyzing the conversion of GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. This sugar nucleotide is the intermediary necessary for the biosynthesis of GDP-D-rhamnose, one of the sugar residues of cepacian, the major exopolysaccharide produced by environmental and human, animal and plant pathogenic isolates of the Burkholderia cepacia complex species. Vmax and Km values of 1.5±0.2 µmol.min−1.mg−1 and 1024±123 µM, respectively, were obtained from the kinetic characterization of the B. cenocepacia J2315 BceN protein by NMR spectroscopy, at 25°C and in the presence of 1 mol MgCl2 per mol of protein. The enzyme activity was strongly inhibited by the substrate, with an estimated Ki of 2913±350 µM. The lack of a functional bceN gene in a mutant derived from B. cepacia IST408 slightly reduced cepacian production. However, in the B. multivorans ATCC17616 with bceN as the single gene in its genome with predicted GMD activity, a bceN mutant did not produce cepacian, indicating that this gene product is required for cepacian biosynthesis.  相似文献   

13.
IIAGlc, the glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system, is an allosteric inhibitor of Escherichia coli glycerol kinase. A linked-functions initial-velocity enzyme kinetics approach is used to define the MgATP-IIAGlc heterotropic allosteric interaction. The interaction is measured by the allosteric coupling constants Q and W, which describe the mutual effect of the ligands on binding affinity and the effect of the allosteric ligand on Vmax, respectively. Allosteric interactions between these ligands display K-type activation and V-type inhibition. The allosteric coupling constant Q is about 3, showing cooperative coupling such that each ligand increases the affinity for binding of the other. The allosteric coupling constant W is about 0.1, showing that the allosteric inhibition is partial such that binding of IIAGlc at saturation does not reduce Vmax to zero. E. coli glycerol kinase is a member of the sugar kinase/heat shock protein 70/actin superfamily, and an element of the superfamily conserved ATPase catalytic core was identified as part of the IIAGlc inhibition network because it is required to transplant IIAGlc allosteric control into a non-allosteric glycerol kinase [A.C. Pawlyk, D.W. Pettigrew, Proc. Natl. Acad. Sci. USA 99 (2002) 11115-11120]. Two of the amino acids at this locus of E. coli glycerol kinase are replaced with those from the non-allosteric enzyme to enable determination of its contributions to MgATP-IIAGlc allosteric coupling. The substitutions reduce the affinity for IIAGlc by about 5-fold without changing significantly the allosteric coupling constants Q and W. The insensitivity of the allosteric coupling constants to the substitutions may indicate that the allosteric network is robust or the locus is not an element of that network. These possibilities may arise from differences of E. coli glycerol kinase relative to other superfamily members with respect to oligomeric structure and location of the allosteric site in a single domain far from the catalytic site.  相似文献   

14.
The number of viable F cells decreases when Escherichia coli recipient cells are mixed with an excess of Hfr cells. Evidence is presented showing that lethal zygosis was accompanied by changes in the physiology of the recipient cells, including (i) inhibition of deoxyribonucleic acid synthesis, (ii) inhibition of β-galactosidase induction, (iii) altered transport and accumulation of galactosides, and (iv) leakage of β-galactosidase into the supernatant fluid. The results are discussed in terms of possible conjugation-associated changes that, at high Hfr to F ratios, lead to lethal zygosis.  相似文献   

15.
16.
The signal-transducing protein EIIAGlc belongs to the phosphoenolpyruvate carbohydrate phosphotransferase system. In its dephosphorylated state, EIIAGlc is a negative regulator for several permeases, including the maltose transporter MalFGK2. How EIIAGlc is targeted to the membrane, how it interacts with the transporter, and how it inhibits sugar uptake remain obscure. We show here that acidic phospholipids together with the N-terminal tail of EIIAGlc are essential for the high affinity binding of the protein to the transporter. Using protein docking prediction and chemical cross-linking, we demonstrate that EIIAGlc binds to the MalK dimer, interacting with both the nucleotide-binding and the C-terminal regulatory domains. Dissection of the ATPase cycle reveals that EIIAGlc does not affect the binding of ATP but rather inhibits the capacity of MalK to cleave ATP. We propose a mechanism of maltose transport inhibition by this central amphitropic regulatory protein.  相似文献   

17.
InEnterobacteriaceae the nonphosphorylated form of IIAG1c of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the Ma1K protein of the maltose transport system, respectively. In this report we show that the IIAGlc-Iike domain of the membrane-bound IIN-acetylglucosamine (IINag) of the PTS can replace IIAGlc in aSalmonella typhimurium crr mutant strain that lacks all soluble IIAGlc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose up take systems. TheStreptococcus thermophilus lactose transporter LacS, which also contains a IIAGlc-like domain, could not replace IIAGlc. Neither IINag nor LacS could replace IIAGlc in activation of adenylate cyclase.  相似文献   

18.
A strain of Synechococcus sp. PCC7942 lacking functional Fe superoxide dismutase (SOD), designated sodB, was characterized by its growth rate, photosynthetic pigments, inhibition of photosynthetic electron transport activity, and total SOD activity at 0°C, 10°C, 17°C, and 27°C in moderate light. At 27°C, the sodB and wild-type strains had similar growth rates, chlorophyll and carotenoid contents, and cyclic photosynthetic electron transport activity. The sodB strain was more sensitive to chilling stress at 17°C than the wild type, indicating a role for FeSOD in protection against photooxidative damage during moderate chilling in light. However, both the wild-type and sodB strains exhibited similar chilling damage at 0°C and 10°C, indicating that the FeSOD does not provide protection against severe chilling stress in light. Total SOD activity was lower in the sodB strain than in the wild type at 17°C and 27°C. Total SOD activity decreased with decreasing temperature in both strains but more so in the wild type. Total SOD activity was equal in the two strains when assayed at 0°C.  相似文献   

19.
A study is presented on the effect of temperature on unidirectional active ion transport, resting electrolyte equilibrium (electrolyte composition), and oxygen consumption in isolated frog skin. The aims were twofold: first, to find out whether the rate of active transport can be changed without affecting the Na+ and K+ balance of skin itself; second, to arrive at minimal ΔNa/ΔO2 values by correlating quantitatively inhibition of active ion transport with inhibition of O2 consumption. NaCl transport was maximal at 20°C. At 28° and at temperatures below 20°, rate of NaCl transport was diminished. In many instances NaCl transport was diminished in skins which maintained their normal Na+ and K+ content. In several cases, however, neither rate of transport nor resting electrolyte equilibrium was affected; in other cases, both were. O2 consumption decreased when lowering the temperature over the range from 28 to 10°C. From a plot of log QOO2 against 1/T an activation energy of µ 13,700 cal. was calculated, valid for the range from 10 to 20°C. It appeared that µ was smaller for temperatures above 20°C. Working between 10 and 20°, it was found that, on the average, 4 to 5 equivalents of Na+ were transported for one mole of O2 consumed in skins with undisturbed resting electrolyte equilibrium.  相似文献   

20.
Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K tam-ch-15  = 8.7 (±0.5)×103 M−1, K tam-ch-100  = 5.9 (±0.4)×105 M−1, K tam-ch-200  = 2.4 (±0.4)×105 M−1 and K hydroxytam-ch-15  = 2.6(±0.3)×104 M−1, K hydroxytam – ch-100  = 5.2 (±0.7)×106 M−1 and K hydroxytam-ch-200  = 5.1 (±0.5)×105 M−1, K endox-ch-15  = 4.1 (±0.4)×103 M−1, K endox-ch-100  = 1.2 (±0.3)×106 M−1 and K endox-ch-200  = 4.7 (±0.5)×105 M−1 with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH2 residues with drug OH and NH2 groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.  相似文献   

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