scholarly journals Functional properties of a sarcoplasmic reticulum Ca2+-ATPase with an altered Ca2+-binding mechanism

1995 ◽  
Vol 309 (2) ◽  
pp. 499-505 ◽  
Author(s):  
F Martinez-Azorin ◽  
F Soler ◽  
J C Gomez-Fernandez ◽  
F Fernandez-Belda
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Equilibrium Constants ◽  
Binding Constants ◽  
Binding Mechanism ◽  
Protein Residues ◽  
Equilibrium Binding ◽  
Positive Cooperativity ◽  
Binding Isotherms ◽  
Fitting Procedures ◽  
Equal Amplitude

Treatment of sarcoplasmic reticulum vesicles with diethylpyrocarbonate in the presence of a large excess of reagent, at pH 6.2 and at room temperature, reveals both a fast- and a slow-reacting population of protein residues. The loss of the Ca(2+)-ATPase activity is mainly associated with the fast-reacting population being partially sensitive to hydroxylamine. There is also an effect on the Ca(2+)-binding mechanism. Shorter derivatization times (5 min) produce a loss of the positive cooperativity of Ca2+ binding. When the treatment was prolonged for 30 min there was an additional decrease in the overall Ca2+ affinity. Curve-fitting procedures applied to the non-cooperative binding isotherms provide the equilibrium constants for the two Ca2+ sites, although they cannot discriminate between interacting and independent site mechanisms. Prestationary kinetics assays show 2 Ca2+:1 ATP ratios, at any extent of Ca2+ saturation, indicating that the Ca2+ sites are not independent. The Ca2+ dissociation profile after derivatization shows a decrease in the dissociation constant for the release of the second Ca2+, which is consistent with interacting sites. Isotopic exchange experiments show fast and slow components of equal amplitude even at subsaturating Ca2+ concentrations, which is incompatible with independent binding sites. The experimental data suggest a modification of the equilibrium binding constants making them more similar, but keeping the interacting character. The structural position of the external (cytoplasmic) and the internal (lumenal) Ca2+ sites remains unaltered in the absence of positive cooperativity.

Improved curve fitting procedures to determine equilibrium binding constants

The Analyst ◽  
2006 ◽  
Vol 131 (10) ◽  
pp. 1145 ◽  
Author(s):  
Frank H. Stootman ◽  
Dianne M. Fisher ◽  
Alison Rodger ◽  
Janice R. Aldrich-Wright
Keyword(s):  
Curve Fitting ◽  
Binding Constants ◽  
Equilibrium Binding ◽  
Fitting Procedures

scholarly journals Architecture of Allosteric Structure. Demonstration of the Fundamental Element of Allosteric Structure of Human Hemoglobin. O2-Equilibrium Binding Curve of Purified Human Hemoglobin in an Effector-Free Supporting Electrolyte.

2019 ◽  
Author(s):  
Francis Knowles
Keyword(s):  
Equilibrium Constants ◽  
Supporting Electrolyte ◽  
Binding Constants ◽  
Fold Increase ◽  
Chloride Ions ◽  
Initial Slope ◽  
Human Hemoglobin ◽  
High Concentration ◽  
Equilibrium Binding ◽  
Oxygen Addition

<p>The O<sub>2</sub>-affinity of human hemoglobin, free of effector molecules, was measured in 0.050 <u>M</u> BisTris, pH 7.0 with HCl, 20<sup>o</sup>C. A Hill plot of O<sub>2</sub>-equilibrium binding data reveals an initial slope of 2 and fails to demonstrate an upward inflection. The tetrameric hemoglobin structure in an effector-free supporting electrolyte can be described as two cooperative dimeric subunits. Equilibrium constants of these steps are related to intrinsic O<sub>2</sub>-binding constants for alpha and beta-chains, K(alpha) and K(beta). The equation of state is comprised of four unknown quantities. O<sub>2</sub>-Binding constants are not expected to be identical for each of the dimeric cooperative subunits. The first cooperative subunit binds O<sub>2</sub> while bound to the second O<sub>2</sub>-free cooperative dimer. The second cooperative dimer binds O<sub>2 </sub>while bound to a fully oxygenated cooperative dimer. Effecter-free human hemoglobin is half saturated with1.9 micromolar oxygen. Addition of 0.1 M NaCl to the supporting electrolyte results in an approximately 4-fold increase in the concentration of O<sub>2</sub> required for half saturation of hemoglobin, 8.1 micromolar oxygen. The mechanism of the response to chloride ions is attributed to neutralization of positively charged residues in the central cavity of Hb<sub>4</sub> by the relatively high concentration of chloride ions. </p>

scholarly journals Architecture of Allosteric Structure. Demonstration of the Fundamental Element of Allosteric Structure of Human Hemoglobin. O2-Equilibrium Binding Curve of Purified Human Hemoglobin in an Effector-Free Supporting Electrolyte.

2019 ◽  
Author(s):  
Francis Knowles
Keyword(s):  
Equilibrium Constants ◽  
Supporting Electrolyte ◽  
Binding Constants ◽  
Fold Increase ◽  
Chloride Ions ◽  
Initial Slope ◽  
Human Hemoglobin ◽  
High Concentration ◽  
Equilibrium Binding ◽  
Oxygen Addition

<p>The O<sub>2</sub>-affinity of human hemoglobin, free of effector molecules, was measured in 0.050 <u>M</u> BisTris, pH 7.0 with HCl, 20<sup>o</sup>C. A Hill plot of O<sub>2</sub>-equilibrium binding data reveals an initial slope of 2 and fails to demonstrate an upward inflection. The tetrameric hemoglobin structure in an effector-free supporting electrolyte can be described as two cooperative dimeric subunits. Equilibrium constants of these steps are related to intrinsic O<sub>2</sub>-binding constants for alpha and beta-chains, K(alpha) and K(beta). The equation of state is comprised of four unknown quantities. O<sub>2</sub>-Binding constants are not expected to be identical for each of the dimeric cooperative subunits. The first cooperative subunit binds O<sub>2</sub> while bound to the second O<sub>2</sub>-free cooperative dimer. The second cooperative dimer binds O<sub>2 </sub>while bound to a fully oxygenated cooperative dimer. Effecter-free human hemoglobin is half saturated with1.9 micromolar oxygen. Addition of 0.1 M NaCl to the supporting electrolyte results in an approximately 4-fold increase in the concentration of O<sub>2</sub> required for half saturation of hemoglobin, 8.1 micromolar oxygen. The mechanism of the response to chloride ions is attributed to neutralization of positively charged residues in the central cavity of Hb<sub>4</sub> by the relatively high concentration of chloride ions. </p>

A Fluorescence Quenching Analysis of the Binding of Fluoroquinolones to Humic Acid

2017 ◽  
Vol 71 (11) ◽  
pp. 2512-2518 ◽  
Author(s):  
Ryan P. Ferrie ◽  
Gregory E. Hewitt ◽  
Bruce D. Anderson
Keyword(s):  
Humic Acid ◽  
Fluorescence Quenching ◽  
Water Solubility ◽  
Binding Constants ◽  
High Water ◽  
Static Quenching ◽  
Temperature Range ◽  
Equilibrium Binding ◽  
Quenching Analysis ◽  
High Water Solubility

Fluorescence quenching was used to investigate the interaction of six fluoroquinolones with humic acid. Static quenching was observed for the binding of ciprofloxacin, enoxacin, fleroxacin, levofloxacin, norfloxacin, and ofloxacin to humic acid. The equilibrium binding constants were found from Stern–Volmer plots of the data. The quenching experiments were repeated over a temperature range of 25–45 ℃ and van’t Hoff plots were generated. From these linear plots, thermodynamic values were calculated for Δ H, Δ G, and Δ S for each of the fluoroquinolones. The equilibrium binding constants were found to be <1 for all the antibiotics studied. The calculated ΔH values were all negative and ranged from −9.5 to −27.6 kJ/mol. The high water solubility of the antibiotics and low ΔH of binding suggests that the antibiotics will be transported easily through the environment. Finally, whether the fluoroquinolones are in a protonated, deprotonated, or partially protonated state is found to correlate to the strength of binding to humic acid.

scholarly journals NmrLineGuru: Standalone and User-Friendly GUIs for Fast 1D NMR Lineshape Simulation and Analysis of Multi-State Equilibrium Binding Models

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chao Feng ◽  
Evgenii L. Kovrigin ◽  
Carol Beth Post
Keyword(s):  
Kinetic Model ◽  
Molecular Interactions ◽  
Rate Constants ◽  
Nmr Spectra ◽  
Equilibrium Constants ◽  
Software Tool ◽  
Biological Interactions ◽  
Equilibrium Binding ◽  
Accurate Quantitative Analysis ◽  
User Friendly

Abstract The ability of high-resolution NMR spectroscopy to readout the response of molecular interactions at multiple atomic sites presents a unique capability to define thermodynamic equilibrium constants and kinetic rate constants for complex, multiple-step biological interactions. Nonetheless, the extraction of the relevant equilibrium binding and rate constants requires the appropriate analysis of not only a readout that follows the equilibrium concentrations of typical binding titration curves, but also the lineshapes of NMR spectra. To best take advantage of NMR data for characterizing molecular interactions, we developed NmrLineGuru, a software tool with a user-friendly graphical user interface (GUI) to model two-state, three-state, and four-state binding processes. Application of NmrLineGuru is through stand-alone GUIs, with no dependency on other software and no scripted input. NMR spectra can be fitted or simulated starting with user-specified input parameters and a chosen kinetic model. The ability to both simulate and fit NMR spectra provides the user the opportunity to not only determine the binding parameters that best reproduce the measured NMR spectra for the selected kinetic model, but to also query the possibility that alternative models agree with the data. NmrLineGuru is shown to provide an accurate, quantitative analysis of complex molecular interactions.

scholarly journals Bifunctional intercalation and sequence specificity in the binding of quinomycin and triostin antibiotics to deoxyribonucleic acid

1978 ◽  
Vol 173 (1) ◽  
pp. 115-128 ◽  
Author(s):  
J S Lee ◽  
M J Waring
Keyword(s):  
Binding Constant ◽  
Binding Constants ◽  
Sequence Specificity ◽  
Peptide Antibiotics ◽  
Naturally Occurring ◽  
Binding Isotherms ◽  
Triostin A ◽  
Specificity Pattern ◽  
Intercalating Agents ◽  
Binding Curves

Quinomycin C, triostin A and triostin C are peptide antibiotics of the quinoxaline family, of which echinomycin (quinomycin A) is also a member. They all remove and reverse the supercoiling of closed circular duplex DNA from bacteriophage PM2 in the fashion characteristic of intercalating drugs, and the unwinding angle at I 0.01 is, in all cases, almost twice that of ethidium. Thus, as with echinomycin, they can be characterized as bifunctional intercalating agents. For the triostins this conclusion has been confirmed by measurements of changes in the viscosity of sonicated rod-like DNA fragments; the helix extension was found to be almost double that expected for a simple monofunctional intercalation process. For triostin A, further evidence for bifunctionality was derived from the cross-over point of binding isotherms to nicked circular and closed circular bacteriophage-PM2DNA. Binding curves for the interaction of quinomycin C and triostin A with a variety of synthetic and naturally occurring nucleic acids were determined by solvent-partition analysis, but triostin C was too insoluble in aqueous solution to make this method applicable. For quinomycin C the highest binding constant was found with Micrococcus lysodeikticus DNA, and its pattern of specificity among natural DNA species was broadly similar to that of echinomycin, although the binding constants were 2–6 times as large. For triostin A the highest binding constant was again found for M. lysodeikticus DNA, but the specificity pattern was quite different from that of the quinomycins. In particular, triostin A bound better to poly(dA-dT) than to the poly(dG-dC) whereas this order was reversed for quinomycin C. There was also evidence that the binding to poly(dA-dT) might be co-operative in nature. No significant interaction could be detected with poly(dA).poly(dT) or with RNA from Escherichia coli. Poly(dG).poly(dC) gave variable results, depending on the source of the polymer. The different patterns of specificity displayed by the quinomycins and triostins are tentatively ascribed to differences in their conformations in solution.

Zur Interaktion von Anthracyclinen und Anthracyclinonen mit DNS / Interaction of Anthracyclines and Anthracyclinones with DNA

1970 ◽  
Vol 25 (4) ◽  
pp. 362-367 ◽  
Author(s):  
H. Berg ◽  
K. Eckardt
Keyword(s):  
Binding Constants ◽  
Cooperative Interaction ◽  
Sugar Residue ◽  
Binding Mechanism ◽  
Buffer Solution ◽  
Binding Ability ◽  
Weak Binding ◽  
Order Of Magnitude ◽  
Dna Complex

It was shown by the polarographic data of the complexes between antracycline antibiotics and DNA that in contrast to their biological inactive aglycones and other antracyclinones, a cooperative interaction of the intercalating chromophore as well as the sugar residue of the anthracycline molecules, is generally responsible for the complex formation with DNA.The high complex binding constants of the antracyclines daunomycin, nogalamycin, galirubin A and galirubin B measured in dimethylsulfoxid-buffer solution are in the same order of magnitude as those of actinomycins. On the other hand, only a weak binding ability of the aglycones daunomycinon, ε-pyrromycinon and aklavinon, as well as of other investigated anthracyclinones and model hydroxyanthraquinones, could be observed.No significant influence of the number and positions of the chromophore hydroxyls could be noticed.The results suggest that the first outerphase addition of the sugar residues to the backbone of the helix gives the necessary space of time for the slower intercalation process of the planar chromophore.In the case of denatured DNA, the antibiotic-DNA-complex has a lowered stability.The important role of the sugar residue for the binding mechanism strongly suggests that modifications of the nature and position of the basic sugar residue should be most valuable for the synthesis of new effective anthracyclines.

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