Kinetic Analysis of a Protein-protein Complex to Determine its Dissociation Constant (KD) and the Effective Concentration (EC50) of an Interplaying Effector Molecule Using Bio-layer Interferometry

BIO-PROTOCOL ◽  
2021 ◽  
Vol 11 (17) ◽  
Author(s):  
Tim Orthwein ◽  
Luciano Huergo ◽  
Karl Forchhammer ◽  
Khaled Selim
Keyword(s):  
Kinetic Analysis ◽  
Dissociation Constant ◽  
Protein Complex ◽  
Effective Concentration ◽  
Effector Molecule ◽  
Dissociation Constant Kd

Erythromycin contracts rabbit colon myocytes via occupation of motilin receptors

1992 ◽  
Vol 262 (1) ◽  
pp. G50-G55 ◽  
Author(s):  
W. L. Hasler ◽  
A. Heldsinger ◽  
O. Y. Chung
Keyword(s):  
Dissociation Constant ◽  
Effective Concentration ◽  
Rabbit Colon ◽  
Colonic Motor ◽  
Half Maximal Effective Concentration ◽  
Gastroduodenal Motility ◽  
Maximal Binding ◽  
Motilin Receptor ◽  
Dissociation Constant Kd

Erythromycin stimulates gastroduodenal motility via action on motilin receptors. We evaluated erythromycin as a colonic muscle motilin agonist using in vitro rabbit colon studies. Isolated myocytes contracted to erythromycin with a half-maximal effective concentration of 2 pM and peak shortening of 22.4 +/- 2.5% at 1 nM, which was superimposable with the response to motilin. 125I-labeled motilin binding to colon muscle homogenates was saturable and specific with a dissociation constant (Kd) of 0.39 nM and maximal binding (Bmax) of 41 +/- 3 fmol/mg protein. Motilin displaced specifically bound 125I-motilin, with a Kd of 0.31 nM. Erythromycin displaced 125I-motilin but was less potent, with an inhibitory constant of 84.0 nM. Bmax values from displacement studies were similar to the Scatchard data. Motilin receptor protection from alkylation by N-ethylmaleimide preserved contraction to motilin and erythromycin but not acetylcholine or cholecystokinin, whereas protection with erythromycin preserved contraction to motilin but not other agonists. In conclusion, erythromycin binds to colon muscle motilin receptors present in densities similar to reported values for the upper gut. Furthermore, erythromycin contracts colonic myocytes via specific action on motilin receptors. Thus erythromycin may have colonic motor-stimulating properties by action on motilin receptors.

scholarly journals Mass Spectrometric Analysis of Antibody—Epitope Peptide Complex Dissociation: Theoretical Concept and Practical Procedure of Binding Strength Characterization

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4776
Author(s):  
Bright D. Danquah ◽  
Kwabena F. M. Opuni ◽  
Claudia Roewer ◽  
Cornelia Koy ◽  
Michael O. Glocker
Keyword(s):  
Mass Spectrometry ◽  
Dissociation Constant ◽  
Gas Phase ◽  
Immune Complex ◽  
Room Temperature ◽  
Electrospray Mass Spectrometry ◽  
Small Sample ◽  
Epitope Peptide ◽  
Ligand Complex ◽  
Dissociation Constant Kd

Electrospray mass spectrometry is applied to determine apparent binding energies and quasi equilibrium dissociation constants of immune complex dissociation reactions in the gas phase. Myoglobin, a natural protein-ligand complex, has been used to develop the procedure which starts from determining mean charge states and normalized and averaged ion intensities. The apparent dissociation constant KD m0g#= 3.60 × 10−12 for the gas phase heme dissociation process was calculated from the mass spectrometry data and by subsequent extrapolation to room temperature to mimic collision conditions for neutral and resting myoglobin. Similarly, for RNAse S dissociation at room temperature a KD m0g#= 4.03 × 10−12 was determined. The protocol was tested with two immune complexes consisting of epitope peptides and monoclonal antibodies. For the epitope peptide dissociation reaction of the FLAG peptide from the antiFLAG antibody complex an apparent gas phase dissociation constant KD m0g#= 4.04 × 10−12 was calculated. Likewise, an apparent KD m0g#= 4.58 × 10−12 was calculated for the troponin I epitope peptide—antiTroponin I antibody immune complex dissociation. Electrospray mass spectrometry is a rapid method, which requires small sample amounts for either identification of protein-bound ligands or for determination of the apparent gas phase protein-ligand complex binding strengths.

scholarly journals Behaviour of neutrophil leucocytes in uniform concentrations of chemotactic factors: contraction waves, cell polarity and persistence

1985 ◽  
Vol 74 (1) ◽  
pp. 75-93
Author(s):  
J.M. Shields ◽  
W.S. Haston
Keyword(s):  
Cell Surface ◽  
Dissociation Constant ◽  
Cell Polarity ◽  
Human Neutrophil ◽  
Cell Movement ◽  
Direct Result ◽  
Surface Receptors ◽  
Chemotactic Peptides ◽  
Chemotactic Factors ◽  
Dissociation Constant Kd

The essential component of any hypothesis of random or directed cell movement is the mechanism of cell polarity. In this paper we describe the polar behaviour of human neutrophil leucocytes in uniform concentrations of chemotactic factors both in suspension and while moving across surfaces. Neutrophils exposed to uniform concentrations of chemotactic factors in suspension around the dissociation constant (Kd) for the receptor rapidly become distinctly bipolar; neutrophils exposed to supraoptimal uniform concentrations (100-fold greater than Kd) of chemotactic factors in suspension, although morphologically active, never reached the same degree of polarity as cells in optimal concentrations. These differences in polarity were shown to be the direct result of equatorial contraction waves stimulated on the cell surface by interaction with chemotactic factors. In optimal concentrations of chemotactic factors, contraction waves were initiated from one region of the cell, whereas in supraoptimal concentrations of chemotactic factors contraction waves emanated from all areas of the cell surface. Asymmetry in the distribution of surface receptors for Fc and C3b were observed in neutrophils polarized in uniform concentrations of chemotactic factor. In neutrophils, motile but not well polarized (in 10(−6) M-N-formylmethionyl-leucyl-phenylalanine (fMLP), receptors were uniformly distributed. In neutrophils polarized in concentrations of fMLP near the Kd for the receptor (10(−8) M) receptors for C3b and Fc were localized in the anterior region of the moving cell. The link between contraction waves, cell polarity and receptor redistribution and their initiation by chemotactic peptides is discussed in the context of neutrophil locomotion and response to chemical signals.

Decreased Reactivity of Plasmin-Streptokinase Towards α-2-Antiplasmin

1979 ◽  
Author(s):  
S Cederholm-Williams ◽  
H Lijnen ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Dissociation Constant ◽  
Active Center ◽  
Rate Constant ◽  
Binding Sites ◽  
Reaction Rate ◽  
Kd Values ◽  
Dissociation Constant Kd

Plasmin (P) is very rapidly (rate constant 2 x 107M-1s-1) inactivated by α-2-antiplasmin (A), and blocking of the lysine binding sites (LSB) of plasmin reduses this rate 50 fold (Eur.J.Biochem.84, 573-578, 1978). Using purified reactants and the plasmin substrate D-Val-Leu-Lys-pNA (S 2251) the influence of streptokinase (SK) on the reaction between P and A was examined. Addition of SK to P reduces the reaction rate with A in a sigmoidal fashion. Assuming the formation of a reversible bimolecular P:SK complex which is unreactive towards A then the dissociation constant Kd of this complex is less than 10-9M. The LSB of P does not play a role in this interaction since similar Kd values are found with modified P, lacking LSB and in the presence of 0.3 mM 6-aminohexanoic acid which blocks the LSB. The P:SK complex is only very slowly inhibited by A with a rate constant < 5 x 102M-1s-1.It is concluded that the α-2-antiplasmin is less reactive towards the plasmin streptokinase complex due to a modification of the active center of plasmin rather than an effect upon the binding sites.

Free cytosolic Ca2+ measured with Ca(2+)-selective electrodes and fura 2 in rat mesenteric resistance arteries

1993 ◽  
Vol 265 (2) ◽  
pp. H741-H746 ◽  
Author(s):  
P. E. Jensen ◽  
M. J. Mulvany ◽  
C. Aalkjaer ◽  
H. Nilsson ◽  
H. Yamaguchi
Keyword(s):  
Dissociation Constant ◽  
Isometric Force ◽  
Force Measurements ◽  
Resistance Arteries ◽  
Fura 2 ◽  
Good Accordance ◽  
Dissociation Constant Kd

Free cytosolic Ca2+ was measured with sub-micrometer-tip, double-barrelled, Ca(2+)-selective electrodes and fura 2 in rat mesenteric resistance arteries. The purpose was to establish intracellular free Ca2+ concentration ([Ca2+]i) values in resting and stimulated vessels. Isolated vessels were mounted for isometric force measurements. Measured with electrodes, mean [Ca2+]i was 115 and 708 nM under resting and norepinephrine-activated conditions, respectively. Fura 2 was calibrated intracellularly including determination of the intracellular dissociation constant (Kd) of the fura 2:Ca2+ complex. The intracellular Kd was 342 nM. With this value of Kd, fura 2 measurements of mean [Ca2+]i were 129 and 537 nM under resting and norepinephrine-activated conditions, respectively. The values measured with the two techniques were thus in good accordance.

Dissociation of the calcium-protein complex of laying hen's plasma

1959 ◽  
Vol 196 (2) ◽  
pp. 371-374 ◽  
Author(s):  
Charles M. Winget ◽  
Arthur H. Smith
Keyword(s):  
Enzymatic Activity ◽  
Dissociation Constant ◽  
Salt Concentration ◽  
Protein Complex ◽  
Calcium Concentration ◽  
Action Principle ◽  
Mass Action ◽  
Shell Formation ◽  
Kinetics Of

The bound, or nondiffusible, calcium component of laying hen's plasma was found to be freely dissociable, in conformance with the mass action principle. No evidence of enzymatic activity, which would dissociate the complex, was found in various tissues and materials associated with shell formation. The kinetics of dissociation of the calcium-protein complex were studied in a series of plasma preparations in which the calcium concentration alone was varied. The dissociation constant for the calcium-protein complex of laying hen's plasma was found to be 10–1.1. Also investigated was the effect of changes in salt concentration (NaCl) and pH on the dissociation of calcium-proteinate.

scholarly journals Binding of NAD+ by cholera toxin

1987 ◽  
Vol 244 (1) ◽  
pp. 225-230 ◽  
Author(s):  
T S Galloway ◽  
S van Heyningen
Keyword(s):  
Binding Site ◽  
Dissociation Constant ◽  
Cholera Toxin ◽  
Equilibrium Dialysis ◽  
Accurate Determination ◽  
Polyacrylamide Gel ◽  
Nad Glycohydrolase ◽  
Competitive Inhibitors ◽  
Dissociation Constant Kd

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.

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