scholarly journals Binding of blood coagulation factor VIII and its light chain to phosphatidylserine/phosphatidylcholine bilayers as measured by ellipsometry

1995 ◽  
Vol 310 (2) ◽  
pp. 539-545 ◽  
Author(s):  
J Spaargaren ◽  
P L A Giesen ◽  
M P Janssen ◽  
J Voorberg ◽  
G M Willems ◽  
...  
Keyword(s):  
Factor Viii ◽  
Dissociation Constant ◽  
Binding Affinity ◽  
Adsorption Kinetics ◽  
Light Chain ◽  
Maximal Surface ◽  
Binding Parameters ◽  
Binding Studies ◽  
Equilibrium Binding ◽  
Maximal Binding

Factor VIII is a plasma protein which plays an essential role in the coagulation system. When assembled with the enzyme Factor IXa on a phospholipid membrane, it functions as a cofactor in the enzyme complex that cleaves the zymogen Factor X to Factor Xa. We studied the binding of both Factor VIII and the Factor VIII light chain to planar phospholipid bilayers consisting of 25% dioleoylphosphatidylserine and 75% dioleoylphosphatidylcholine (PSPC) by ellipsometry. Equilibrium-binding studies revealed that both Factor VIII and its light chain bind with high affinity to PSPC bilayers. The binding affinity of Factor VIII, with a dissociation constant Kd of 0.24 nM, was comparable with that of the Factor VIII light chain (Kd 0.49 nM). Maximal binding was 2.3 mmol of protein per mol of PSPC for Factor VIII and 7.1 mmol of protein per mol of PSPC for the Factor VIII light chain. Adsorption kinetics of both Factor VIII and its light chain conformed to the classical Langmuir adsorption model yielding dissociation constants calculated from the rates of adsorption that were similar to those obtained by equilibrium-binding studies. In contrast, measurements of rates of desorption revealed a deviation from those expected for a single class of binding sites. The desorption rate of Factor VIII increased with increasing residence time on the lipid membrane. This indicates transition of Factor VIII to a configuration with a lower binding affinity. As this time-dependent change in affinity could affect the validity of the measurement of binding parameters, in particular equilibrium-binding determinations carried out on a long timescale, binding affinity was also estimated from adsorption kinetics at half-maximal surface coverage, a relatively rapid procedure for the determination of the affinity. A Kd of 0.087 nM was obtained under these conditions. Measurement of equilibrium binding to small PSPC vesicles, a system in which equilibrium is rapidly attained, resulted in similar binding parameters (Kd = 0.13 nM and a maximal binding of 2.8 mmol of protein per mol of PSPC). These data confirm the results of equilibrium binding to planar bilayers. Taken together, our results indicate that Factor VIII, by means of its 80 kDa light chain, binds to PSPC bilayers with a dissociation constant below the concentration of Factor VIII in plasma and therefore may readily bind to exposed phospholipid membranes under physiological conditions.

The preparation and phospholipid binding property of the C2 domain of human factor VIII

2003 ◽  
Vol 89 (05) ◽  
pp. 788-794 ◽  
Author(s):  
Kazuya Takeshima ◽  
Christina Smith ◽  
Jonathan Tait ◽  
Kazuo Fujikawa
Keyword(s):  
Factor Viii ◽  
Binding Affinity ◽  
Light Chain ◽  
Human Factor ◽  
Binding Activity ◽  
Exchange Chromatography ◽  
Binding Property ◽  
C2 Domain ◽  
Phospholipid Binding ◽  
Human Factor Viii

SummaryThe C2 domain of human factor VIII was expressed in a yeast secretion system and its binding properties were studied. A cDNA coding the C2 domain sequence of human factor VIII with a N-terminal six amino acids extension (C-C2) was constructed, transformed into Pichia pastoris cells and expressed. The product was purified by ammonium sulfate fractionation and anion exchange chromatography. It emerged as a single peak from both ion exchange and gel filtration columns, indicating C-C2 is a homogenous monomer. The binding activity of C-C2 to phosphatidylserine-containing phospholipid vesicles was measured by competitive binding with annexin V. The values of IC50were approximately 70nM for both factor VIII and its light chain, but were about 7000nM for C-C2. These results indicated C-C2 has 100-fold less binding affinity than factor VIII or the light chain. Direct binding to solidified phosphatidylserine-containing phospholipids also showed that C-C2 has ~50-fold less binding affinity than does the light chain. C-C2 poorly inhibited Xase activity. These results together clearly show that the C2 domain alone does not have full membrane binding activity, and suggest that the other light chain domains, A3 and/or C1, are also involved in the phospholipid binding activity of factor VIII.

Equilibrium and kinetic studies of the interaction of site-specific ligands with acetylcholinesterase from Electrophorus electricus

1978 ◽  
Vol 56 (12) ◽  
pp. 1133-1140 ◽  
Author(s):  
George Tomlinson ◽  
Bulent Mutus ◽  
W. John Rutherford
Keyword(s):  
Steady State ◽  
Dissociation Constant ◽  
Kinetic Studies ◽  
Order Rate Constant ◽  
Competitive Inhibitor ◽  
Binding Studies ◽  
Electrophorus Electricus ◽  
Equilibrium Binding ◽  
Low Ionic Strength ◽  
Hydrolysis Of

The interactions of edrophonium chloride, gallamine triiodide, and propidium diiodide with affinity-purified acetylcholinesterase from Electrophorus electricus have been examined under conditions of low ionic strength (0.001 M Tris, pH 8.0) using kinetic and fluorescence titration techniques. Edrophonium is a competitive inhibitor of the steady-state hydrolysis of acetylthiocholine, with an inhibition constant, Kcomp, of 1.2 × 10−8 M. Double reciprocal plots in the presence of either gallamine or propidium are nonlinear. Similarly, the pre-steady-state carbamoylation of the enzyme by 7-(dimethylcarbamoyloxy)-N-methyl quinolinium iodide is competitively inhibited by edrophonium, whereas the intercepts of the double reciprocal plots of pseudo-first-order rate constant of carbamoylation versus substrate concentration are displaced downwards in the presence of gallamine or propidium. These results, and those of equilibrium binding studies utilizing the fluorescence properties of bound propidium, suggest that gallamine and propidium compete for a peripheral class of anionic sites on the enzyme, whereas edrophonium binds to the anionic subsite of the catalytic site. The characteristics of propidium binding to the eel enzyme differ from those previously observed with enzyme isolated from Torpedo californica. Whereas the tetrameric Torpedo enzyme possesses four binding sites of equal affinity for propidium, the eel enzyme appears to have two classes of propidium binding site. One set of approximately two sites per tetramer is characterized by a dissociation constant of approximately 2–5 × 10−8 M; a second set of two sites bind propidium with a dissociation constant of 4 × 10−6 M. Possible reasons for these differences are discussed.

scholarly journals Equilibrium-binding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions

1980 ◽  
Vol 185 (1) ◽  
pp. 107-114 ◽  
Author(s):  
I A Nieduszynski ◽  
J K Sheehan ◽  
C F Phelps ◽  
T E Hardingham ◽  
H Muir
Keyword(s):  
Dissociation Constant ◽  
Equilibrium Dialysis ◽  
Guanidine Hydrochloride ◽  
Dilute Solution ◽  
Binding Studies ◽  
Laryngeal Cartilage ◽  
Equilibrium Binding ◽  
Cartilage Proteoglycans ◽  
Dissociation Constant Kd

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 × 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 × 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol.wt. 670 000), an average Kd of 12 × 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.

Plasminogen-Plasmin System IX. Specific Binding of Tranexamic Acid to Plasmin

1975 ◽  
Vol 33 (03) ◽  
pp. 573-585 ◽  
Author(s):  
Masahiro Iwamoto
Keyword(s):  
Tranexamic Acid ◽  
Affinity Chromatography ◽  
Dissociation Constant ◽  
Factor Xiii ◽  
Specific Binding ◽  
The Other ◽  
Inhibitory Mechanism ◽  
Binding Studies ◽  
Similar Affinity ◽  
Fibrin Structure

SummaryInteractions between tranexamic acid and protein were studied in respect of the antifibrinolytic actions of tranexamic acid. Tranexamic acid did neither show any interaction with fibrinogen or fibrin, nor was incorporated into cross-linked fibrin structure by the action of factor XIII. On the other hand, tranexamic acid bound to human plasmin with a dissociation constant of 3.5 × 10−5 M, which was very close to the inhibition constant (3.6 × 10−5 M) for this compound in inhibiting plasmin-induced fibrinolysis. The binding site of tranexamic acid on plasmin was not the catalytic site of plasmin, because TLCK-blocked plasmin also showed a similar affinity to tranexamic acid (the dissociation constant, 2.9–4.8 × 10−5 M).In the binding studies with the highly purified plasminogen and TLCK-plasmin preparations which were obtained by affinity chromatography on lysine-substituted Sepharose, the molar binding ratio was shown to be 1.5–1.6 moles tranexamic acid per one mole protein.On the basis of these and other findings, a model for the inhibitory mechanism of tranexamic acid is presented.

Enhancement of Plasminogen Binding to U937 Cells and Fibrin by Complestatin

1997 ◽  
Vol 77 (01) ◽  
pp. 137-142 ◽  
Author(s):  
Kiyoshi Tachikawa ◽  
Keiji Hasurni ◽  
Akira Endo
Keyword(s):  
Binding Site ◽  
Binding Affinity ◽  
Blood Cells ◽  
Aminocaproic Acid ◽  
U937 Cells ◽  
Biochemical Basis ◽  
Equilibrium Binding ◽  
Pharmacological Stimulation ◽  
Endogenous Fibrinolysis ◽  
Stimulation Of

SummaryPlasminogen binds to endothelial and blood cells as well as to fibrin, where the zymogen is efficiently activated and protected from inhibition by α2-antiplasmin. In the present study we have found that complestatin, a peptide-like metabolite of a streptomyces, enhances binding of plasminogen to cells and fibrin. Complestatin, at concentrations ranging from 1 to 5 μM, doubled 125I-plasminogen binding to U937 cells both in the absence and presence of lipoprotein(a), a putative physiological competitor of plasminogen. The binding of 125I-plasminogen in the presence of complestatin was abolished by e-aminocaproic acid, suggesting that the lysine binding site(s) of the plasminogen molecule are involved in the binding. Equilibrium binding analyses indicated that complestatin increased the maximum binding of 125I-plasminogen to U937 cells without affecting the binding affinity. Complestatin was also effective in increasing 125I-plasminogen binding to fibrin, causing 2-fold elevation of the binding at ~1 μM. Along with the potentiation of plasminogen binding, complestatin enhanced plasmin formation, and thereby increased fibrinolysis. These results would provide a biochemical basis for a pharmacological stimulation of endogenous fibrinolysis through a promotion of plasminogen binding to cells and fibrin.

Bronchospasmolytic and Adenosine Binding Activity of 8- (Proline / Pyrazole)-Substituted Xanthine Derivatives

2020 ◽  
Vol 17 ◽  
Author(s):  
Sneha Singh ◽  
Madhwi Ojha ◽  
Divya Yadav ◽  
Sonja Kachler ◽  
Karl-Norbert Klotz ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Radioligand Binding ◽  
Binding Activity ◽  
Receptor Subtypes ◽  
Significant Protection ◽  
Binding Studies ◽  
Standard Drug ◽  
Xanthine Derivatives ◽  
Radioligand Binding Studies ◽  
Micromolar Range

Background: ABSTRACT: Background: 8-Phenyltheophylline derivatives exhibit prophylactic effects at a specific dose but do not produce the cardiovascular or emetic side effects associated with xanthines, thereby exhibiting unique characteristics of potential therapeutic importance. Methods: Novel series of 8-(proline/pyrazole)-substituted xanthine analogs has been synthesized. The affinity and selectivity of compounds to adenosine receptors have been assessed by radioligand binding studies. The synthesized compounds also showed good bronchospasmolytic properties (increased onset of bronchospasm; decreased duration of jerks) with 100% survival of animals in comparison to the standard drug. Besides, compound 8f & 9f showed good binding affinity in comparison to other synthesized compounds in the micromolar range. Results: The maximum binding affinity of these compounds was observed for A2B receptors, which is ~ 7 or 10 times higher as compared to A1, A2A and A3 receptors. The newly synthesized derivatives 8f, 9a-f, 17g-m, and 18g-m displayed significant protection against histamine aerosol induced bronchospasm in guinea pigs. Conclusion: Newly synthesized proline/pyrazole based xanthines compounds showed a satisfactory binding affinity for adenosine receptor subtypes. Replacement or variation of substituted proline ring with substituted pyrazole scaffold at 8thposition of xanthine moiety resulted in the reduction of adenosine binding affinity and bronchospasmolytic effects.

scholarly journals Kinetic mechanism of Na+-coupled aspartate transport catalyzed by GltTk

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Gianluca Trinco ◽  
Valentina Arkhipova ◽  
Alisa A. Garaeva ◽  
Cedric A. J. Hutter ◽  
Markus A. Seeger ◽  
...  
Keyword(s):  
Kinetic Mechanism ◽  
Protein Quantification ◽  
Detergent Solution ◽  
Sodium Ions ◽  
Binding Studies ◽  
Equilibrium Binding ◽  
Uptake Rates ◽  
Bona Fide ◽  
Inside Out

AbstractIt is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified GltTk, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on GltPh using this equation allowed for determination of the turnover number (0.14 s−1), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented GltTk proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging.

A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

scholarly journals The determination of binding parameters when the total and free substrate concentrations are not approximately equal

1979 ◽  
Vol 179 (3) ◽  
pp. 697-700 ◽  
Author(s):  
N Gains
Keyword(s):  
Substrate Concentration ◽  
Graphical Method ◽  
Enzyme Concentration ◽  
Binding Parameters ◽  
Free Concentration ◽  
Equilibrium Binding ◽  
Substrate Concentrations

By using a standard graphical method values of Km and V may be found that are independent of the conditions and assumptions that the total substrate concentration approximates to its free concentration and that Km is much larger than the enzyme concentration. The procedure is also applicable to the determination of equilibrium binding parameters of a ligand to a macromolecule.

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