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1.
The conformational change in a single molecular species, beta3, of beta-conglycinin in an acidic ethanol solution was kinetically studied by the stopped-flow technique, utilizing the intrinsic fluorescence of proteins and the fluorescence of 1-anilinonaphthalene-8-sulfonic acid (ANS) bound to the proteins. The time-course of the intrinsic fluorescence changes clearly showed the rate of conformational change below and above 25% ethanol to be quite different from each other. ANS could bind well to the protein in an ethanol concentration range of 15-25%. However, the rate of conformational change of the protein corresponding to that for ANS binding could not be obtained at less than 25% ethanol, while the rate of conformational change agreed well with that for ANS binding at more than 25% ethanol. In addition, the process showing the greatest and slowest ANS binding was not apparent in the denaturation of beta-conglycinin under the conditions employed. These results lead to the conclusions that the beta-conglycinin structure could be maintained in the mild molten globule-like denaturation state, and that various tertiary structural changes could take place without any significant effect on the high sensitivity of intrinsic fluorescence after the secondary structural changes.  相似文献   

2.
Experiments are presented which show that oxaloacetate and analogs thereof with (R)-malate substructure, on interaction with citrate synthase linked to synthase 8-anilinonaphthalene 1-sulfonate (ANS), induce identical conformational changes of a characteristic magnitude. A conformational change of lower magnitude is also produced on binding of CoASH or ATP to citrate synthase.ANS and is completed on addition of oxaloacetate. The significance of these ligand-dependent conformational changes is discussed.  相似文献   

3.
The binding of 8-anilino-1-naphthalenesulfonate (ANS) to ciliary dynein ATPase leads to a marked increase in the dye's fluorescence intensity, accompanied by a blue shift in the observed fluorescence emission maximum. We found that dynein has 37 +/- 3 ANS binding sites and that experimentally applied ANS concentrations failed to alter enzyme activity. The fluorescence properties of the enzyme-dye complex were used to learn more about the binding characteristics of dynein substrates and effectors and to probe for possible conformational changes of the enzyme. The fluorescence of the dynein-ANS complex is increased by a number of substrates, including ATP, GTP, and UTP. The transfer of excitation energy from dynein chromophores to adsorbed ANS was also investigated. Our findings indicate that dynein appears to undergo a localized conformational change in its interaction with ATP. Native dynein was also found to be conformationally different from heat-activated or NEM-modified enzyme as evidenced by the emission and excitation spectra of the various enzyme-ANS complexes.  相似文献   

4.
The effect of NAD on the binding of 1-anilino-8-naphthalene sulfonate (ANS) to yeast glyceraldehyde-3-phosphate dehydrogenase has been studied using difference spectrophotometric and fluorescence techniques. Coenzyme addition causes the displacement of ANS from its complex with the dehydrogenase, as suggested by the effect of NAD on the fluorescence of the enzyme--ANS complex, as well as on the magnitude of the difference spectrum of the complex. Adenine containing NAD fragments, adenosine, 5'-AMP, and ADP were shown to compete with ANS for the common site on the enzyme using fluorimetric technique; in the case of adenosine and 5'-AMP a direct method of analytical ultracentrifugation was also employed. The results obtained by both methods suggest the dye binding at the adenine subsite of the dehydrogenase. The interaction with ANS causes no detectable conformational changes of the protein. The fluorescence of the dye-enzyme complex increases and the emission maximum shifts to shorter wavelengths on addition of nicotinamide mononucleotide. This suggest some conformational changes to occur in the microenvironment of the bound dye in response to the interaction with the ligand in the nicotinamide subsite. The participation of the nicotinamide subsite of the active center in determining the character of conformational transitions associated with coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase is discussed.  相似文献   

5.
We have tested the working hypothesis that anesthetics, by labilizing lipid-protein interactions, induce conformational changes in membrane proteins involved in the transmission of neural impulses. In the first communication of this series we report that general anesthetics induce changes in the fluorescence of the probes ANS and NPN in model membranes, lipid vesicles and mitochondria. The changes observed concern the quantumyield but not the position of the emission maximum. Such changes may be interpreted as due to fluidization of the membrane core (NPN), accompanied by variable effects in the membrane surface(ANS).  相似文献   

6.
An immune response was induced in vivo on C3H/He ♂ mouse strain with Bovine Serum Albumin (BSA), or Sheep Red Blood Cells (SRBC). The membrane fluorescence changes of activated splenic lymphocytes were studied two weeks after the injection of antigens. Experiments were performed with the hydrophobic fluorescent probe: 1-anilino-8-naphthalene sulphonate (ANS). The kinetic studies further indicated that the course of fluorescence changes may considerably vary depending on antigens. Their fluorescence intensities were lower than control values. A maximum decrease of fluorescence was recorded on days 1, 6 and 9 after immunization with BSA-stimulated lymphocytes. SRBC-stimulated lymphocytes exhibited a maximum ANS fluorescence decrease on days 4 and 9 after immunization. These fluorescence phenomena would be in an inverse relationship with the electrokinetic surface potential of activated lymphocytes, as assessed by the electrophoretic mobility analysis (EPM). Some parameters affecting the ANS fluorescence in T and B cells are discussed. Quantification of hydrophobic sites in splenic cells would indicate that forces other than the hydrophobic ones may also be involved in the dye-binding changes following immune activation.  相似文献   

7.
A proximate method for conformational changes detection in immunoglobulin G preparations used in research and medical practice is introduced. This method is based on the measurement of IgG-connected 1,8-anilinenaphtalenesulphonate fluorescent probe (ANS) time of damping. When the protein structure is broken fluorescence damping time of IgG-connected ANS rises up.  相似文献   

8.
The fluorescent probe 8-anilino-1-naphthalene sulphonic acid (ANS) has been used to demonstrate the accessibility of the heme of cytochrome c — mixed mitochondrial phospholipid complexes to the solvent. Contrary to earlier reports fluorescence techniques using ANS do not detect redox induced conformational changes of these complexes.  相似文献   

9.
The interaction between C1q and immune complexes is inhibited by 1-anilino-8-naphthalenesulfonate (ANS) in the concentration range of 2-4 mM. ANS binds to Clq with a 20-fold higher affinity than to IgG [(1986) Mol. Immunol. 23, 39-44] and therefore it is possible to label only C1q with ANS in the presence of IgG. Under such conditions no inhibition is observed. Addition of monomer IgG to a solution of C1q-bound ANS did not significantly alter the fluorescence of the ANS. However when oligomeric IgG was added there was a 2-fold increase in fluorescence over the same IgG concentration range. When C1q was pretreated with diethylpyrocarbonate there was little change in the fluorescence when IgG oligomers were added to C1q:ANS solutions. These results suggest that C1q undergoes conformational changes upon binding to IgG oligomers.  相似文献   

10.
The in vivo effect of Concanavalin A (Con A), or bacterial lipopolysaccharide (LPS) on mouse spleen cell populations was investigated. The membrane fluorescence changes of activated splenic lymphocytes were studied during two weeks after the injection of polyclonal immunogens. Experiments were performed with the hydrophobic fluorescent probe: 1-anilino-8-naphthalene sulphonate (ANS). The kinetic studies further indicated that the course of fluorescence changes may considerably vary depending on both immunogens. These fluorescence intensity changes would be in direct relation to the electrokinetic surface potential changes of activated lymphocytes, as assessed by the electrophoretic mobility analysis. By comparison with the inverse relationship observed in our previous study, it would be concluded that the relation (direct or inverse) between ANS fluorescence and electrokinetic potential depends on the net electrical charge of the antigens used.  相似文献   

11.
1-anilino-8-naphtalenesulfonate (ANS) is a hydrophobic dipole previously used to demonstrate that the proton for potassium exchange by the gastric HK-ATPase is electroneutral. In this paper, we demonstrate that ANS binds to gastric membranes and probes conformational changes of the HK-ATPase independently of any active H for K exchange. Conformational changes require the presence of potassium-valinomycin and are not triggered by sodium. Potassium effect is enhanced by ATP, in the presence and in the absence of magnesium and, by ADP, in the presence of magnesium. Labeling of the pig HK-ATPase K518 by fluorescein-5-isothiocyanate inhibits the enzyme activity and knocks out the ATP effect on ANS fluorescence. Scherring 28080 and the monoclonal antibody 95-111, two competitive inhibitors of K-activated ATPase dephosphorylation, do not modify K-effect on ANS fluorescence but inhibit ATP effects. This supports that ANS does not probe K-site between the H1–H2 loop. Treatment of gastric membranes with trypsin does not inhibit the ANS response to potassium but does inhibit the response to ATP. This suggests that the ATP site inducing the ANS response is cytoplasmic and the potassium site is intramembranous. Titration reveals that one mole of ANS interacts with one mole of ATPase. We suggest that ANS probes a hydrophobic potassium site of gastric ATPase and that addition of ATP and ADP-Mg embed that site. Received: 16 July 1997/Revised: 10 June 1998  相似文献   

12.
The acid-induced isomerization (the N-F transition) and expansion of bovine plasma albumin--1-anilino-8-naphthalenesulfonate complex, BPA-ANS1.0 complex (molar ratio of added ANS to BPA = 1.0) were studied by measuring fluorescence and induced CD spectra of ANS. Decrease in the reciprocal of fluorescence polarization, increase in fluorescence intensity and blue shift of fluorescence of ANS in BPA-ANS1.0 complex were correlated with the initial part of the N-F transition and/or the N-F1 transition. Induced CD spectra of ANS showed positive bands at 250-258 and 320-350 nm and one negative band at 280 nm. Most of changes (decreases) in -[theta]280 were also correlated with the initial part of the N-F transition and/or the N-F1 transition. Changes in fluorescence parameters and induced CD spectra of ANS (-[theta]280) might indicate the conformational changes around a strong ANS binding site in the N-terminal domain (Reed et al. (1975), Jonas & Weber (1970) and Brown & Shockley (1982].  相似文献   

13.
Interaction of phosphorylase with 8-anilino-1-naphthalene-sulfonate (ANS) results in the formation of an ANS-protein complex. The microenvironment of the protein-bound dye changes depending on pH. Using fluorimetric titration, the dissociation constants for the complex (Kd = 23 and 57 microM for pH 6.2 and 6.8, respectively) were determined. The mode of the enzyme inhibition by ANS also changes depending on pH. At pH 6.8, ANS competitively inhibits the enzyme with respect to AMP, but does not compete with the nucleotide at pH 6.2; the corresponding Ki values are equal to 160 and 26 microM. The protective effect of ligands from the inhibiting effect of ANS was studied. It was shown that at pH 6.2, the enzyme is protected from the inhibition only by the substrate, glucose-1-phosphate, whereas at pH 6.8--by the allosteric inhibitor, glucose-6-phosphate. These findings suggest that at pH 6.2 the conformation of the enzyme molecule is induced by the substrate, while at pH 6.8--by the allosteric inhibitor. ANS binding in the vicinity of the active or allosteric centers is due to the pH-dependent conformational transition. The data obtained suggest that the pH changes within the range of 6.2-6.8 are essential for the regulation of enzyme activity.  相似文献   

14.
The interaction between 1-anilino-8-naphthalenesulfonate (ANS) and yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) and the use of ANS as a probe for studying the structure and function of phosphoglycerate kinase has been investigated. The interaction has been studied by kinetic methods, equilibrium dialysis, and fluorometric titrations. ANS inhibits the activity of the enzyme. More than one inhibitor site exists. ANS is competitive with MgATP and noncompetitive with 3-phosphoglycerate at the first detected inhibitor binding site. The Ki value is 1-2 mM. Several ANS molecules bind to the enzyme. By fluorometric titrations the first detected site has a dissociation constant that is in the same range as Ki or bigger. When ANS interacts with phosphoglycerate kinase its fluorescence is increased and a blue shift occurs. ANS appears to bind to a strongly hydrophobic site. The fluorescence is sensitive to the addition of substrates. ADP, ATP, or combinations of Mg2+ and nucleotide decreases the fluorescence as does free Mg2+. 3-Phosphoglycerate, on the other hand, increases the fluorescence giving evidence for conformational changes upon 3-phosphoglycerate binding.  相似文献   

15.
The effect of oxidized dithiothreitol (DTT) on the conformation and function of arginine kinase from shrimp Feneropenaeus chinensis was investigated with the methods of intrinsic fluorescence, ANS fluorescence, size exclusion chromatography (SEC), sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), and activity assay. The excess molecular oxidized dithiothreitol could result in a loss of activity and conformational change of arginine kinase. The oxidized arginine kinase was characterized by monitoring the changes of fluorescence emission wavelength (excitation wavelength: 295 nm) and the intensity of 1-anilino-8-naphthalenesulfonate (ANS) binding (excitation wavelength: 380 nm) to the protein. The results of fluorescence spectra showed that the presence of oxidized DTT could result in a marked change in the enzyme tertiary structure. The conformational changes of native and oxidized arginine kinase are induced by the presence of the full set of transition state analog (TSA) components. The results of size exclusion chromatography and SDS-PAGE indicated that no disulfide bond was formed among the protein molecules in the oxidized-DTT solution.  相似文献   

16.
Proteins for therapeutic use may contain small amounts of partially misfolded monomeric precursors to postproduction aggregation. To detect these misfolded proteins in the presence of an excess of properly folded protein, fluorescent probes such as 8-anilino-1-naphthalene sulfonate (ANS) are commonly used. We investigated the possibility of using isothermal titration calorimetry (ITC) to improve the detection of this type of conformational change using hydrophobic probes. As a case study, conformational changes in human polyclonal immunoglobulin G (IgG) were monitored by measuring the enthalpies of binding of ANS using ITC. Results were compared with those using fluorescence spectroscopy. IgG heated at 63 °C was used as a model system for “damaged” IgG. Heat-treated IgG can be detected already at levels below 5% with both ITC and fluorescence. However, ITC allows a much wider molar probe-to-protein ratio to be sampled. In particular, using reverse titration experiments (allowing high probe-to-protein ratios not available to fluorescence spectroscopy), an increase in the number of binding sites with a Kd > 10 mM was observed for heat-treated IgG, reflecting subtle changes in structure. Both ITC and fluorescence spectroscopy showed low background signals for native IgG. The nature of the background signals was not clear from the fluorescence measurements. However, further analysis of the ITC background signals shows that a fraction (8%) binds ANS with a dissociation constant of approximately 0.2 mM. Measurements were also carried out at pH 4.5. Precipitation of IgG was induced by ANS at concentrations above 0.5 mM, interfering with the ITC measurements. Instead, with the nonfluorescent probes 4-amino-1-naphthalene sulfonate and 1-naphthalene sulfonate, no precipitation is observed. These probes yield differences in the enthalpies of binding to heated and nonheated IgG similar to ANS. The data illustrate that ITC with low-molecular-weight probes is a versatile tool to monitor conformational changes in proteins with a wider application potential than fluorescence measurements.  相似文献   

17.
The effect of adrenochrome semicarbazide on the conformation of erythrocyte ghost membranes has been studied by ANS fluorescence, lipid and sulfhydryl spin labels and circular dichroism. No large conformational alterations in the membrane were detected by these techniques. Noncompetitive quenching of ANS fluorescence by ADCS suggests ADCS to interact with the membrane at sites close to the ANS binding domain.  相似文献   

18.
Calmodulin has been shown to alter its conformation so as to interact with a number of target proteins upon Ca2+ binding. A Ca2(+)-binding study of calmodulin was performed by monitoring the fluorescence of intrinsic tyrosine residues and the probe 1-anilinonaphthalene-8-sulfonate (ANS). ANS fluorescence was shown to reflect Ca2+ binding to both high- and low-affinity sites. On the one hand, tyrosine fluorescence was sensitive only to the high-affinity Ca2+ binding. Temperature-jump investigation of the ternary complex of Ca2(+)-calmodulin-ANS in combination with monitoring of ANS fluorescence demonstrated the kinetic characteristics of the conformational change. The relaxation process was attributed to Ca2(+)-induced conformational change and the rate constants of this process were evaluated. On the basis of the rate constants of the conformational change, a rapid response of calmodulin in Ca2+ signaling is suggested.  相似文献   

19.
Several recent reports indicate that patients with Huntington's Disease (HD) may manifest membrane abnormalities in a wide variety of cells including peripheral blood lymphocytes. In this study, flow cytometry is used in conjunction with the fluorescent membrane probe, 8-anilino-1-naphthalene sulfonate (ANS), to examine peripheral blood lymphocytes from 16 HD patients and 14 age- and diet-matched control subjects. Increased ANS fluorescence intensity of lymphocytes (p less than 0.02) was found in HD patients as compared to control subjects. These differences are masked when the mean fluorescence of the total leukocyte population is measured, possibly explaining conflicting data of other investigators. These observed differences in ANS fluorescence intensity between HD patients and control subjects support the concept of a gene defect which may be expressed as membrane alterations in non-neural as well as neural cells. The selective alterations of lymphocytes may also reflect altered immunological activity reported in HD.  相似文献   

20.
Using steady-state, polarized, and phase-modulation fluorometry, the dithiothreitol-induced denaturation of insulin and formation of its complex with alpha-crystallin in solution were studied. Prevention of the aggregation of insulin by alpha-crystallin is due to formation of chaperone complexes, i.e. interaction of chains of the denatured insulin with alpha-crystallin. The conformational changes in alpha-crystallin that occur during complex formation are rather small. It is unlikely that N-termini are directly involved in the complex formation. The 8-anilino-1-naphthalenesulfonate (ANS) is not sensitive to the complex formation. ANS emits mainly from alpha-crystallin monomers, dimers, and tetramers, but not from oligomers or aggregates. The possibility of highly sensitive detection of aggregates by light scattering using a spectrofluorometer with crossed monochromators is demonstrated.  相似文献   

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