scholarly journals Binding of α-thrombin to fibrin depends on the quality of the fibrin network

1994 ◽  
Vol 298 (1) ◽  
pp. 157-163 ◽  
Author(s):  
H Bänninger ◽  
B Lämmle ◽  
M Furlan
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Scatchard Analysis ◽  
Binding Parameters ◽  
Irreversible Binding ◽  
Binding Data ◽  
Fibrin Network ◽  
Straight Lines ◽  
Fold Excess

Binding of human alpha-thrombin to fibrin was studied in a purified system at pH 7.35, I 0.08 and 37 degrees C. Binding experiments with active thrombin resulted in fibrin clots of variable quality, depending on the thrombin concentration: opaque gels composed of ‘coarse’ network were produced at low thrombin concentrations, while increasing concentrations of thrombin led to more translucent ‘fine’ gels. Scatchard analysis showed a non-linear dependence of thrombin binding to fibrin, suggesting the existence in fibrin(ogen) of multiple classes of binding sites for thrombin. Binding of catalytic-site-inhibited thrombin was investigated in clots of defined quality produced with three different concentrations of a thrombin-like enzyme, batroxobin (EC 3.4.21.29). Straight lines of different slopes were established by Scatchard analysis of binding data at each fixed batroxobin concentration. These results favour a model according to which binding affinity for thrombin depends on the thickness of fibrin bundles. Labelled active-site-inactivated thrombin incorporated in batroxobin-induced clots was only sparingly released during incubation for 24 h in the presence of a 200-fold excess of unlabelled thrombin, indicating that thrombin binding to fibrin is not reversible and that Scatchard analysis is not appropriate for quantification of binding parameters. Irreversible binding of thrombin appears to reflect trapping of thrombin molecules within fibrin fibres. The amount of trapped thrombin depends on the quality of the fibrin fibres, which in turn is determined by the concentration of the clotting enzyme.

scholarly journals Hepoxilin A3-specific binding in human neutrophils

1996 ◽  
Vol 313 (2) ◽  
pp. 537-541 ◽  
Author(s):  
Denis REYNAUD ◽  
Peter DEMIN ◽  
Cecil R. PACE-ASCIAK
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Leukotriene B4 ◽  
Proteinase K ◽  
Human Neutrophils ◽  
Scatchard Analysis ◽  
Intact Cells ◽  
Specific Binding Sites ◽  
Binding Data ◽  
Hepoxilin A3

Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 °C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 °C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3±9.1 nM and 8.86±1.4 pmol/ml per 2×106 cells (±S.E.M.) respectively reflecting approx. 2.67×106 sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.

scholarly journals Calcium Binding Sites on Human Blood Platelets

1977 ◽  
Author(s):  
S. Heptinstall
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Blood Platelets ◽  
Human Platelets ◽  
Extracellular Calcium ◽  
Scatchard Analysis ◽  
Magnesium Ions ◽  
Binding Data ◽  
Calcium Binding Sites ◽  
Human Blood Platelets

Extracellular calcium ions are required for platelets to aggregate in response to various aggregating agents. Although magnesium ions can sometimes stimulate aggregation they only do so when a small amount of calcium is present. The calcium bound to washed human platelets suspended in buffered saline containing 0-200μM+5CaCl2 depends upon the extracellular calcium concentration. Scatchard analysis of the binding data suggests that a few (0,8 × 106) relatively high affinity (K = 85,000) calcium binding sites are present on each platelet. When 2.5mM MgCl2 is included in the saline suspensions the calcium bound to the platelets is only reduced at the higher calcium concentrations. Magnesium ions do not displace the tightly bound calcium. It is suggested that these specific calcium binding sites are involved in platelet aggregation.

The Binding Of 125I-Von Willebrand Factor To Human Platelets In The Presence Of Ristocetin

1981 ◽  
Author(s):  
P Silber ◽  
T H Finlay
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Binding Constant ◽  
Specific Binding ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
Binding Data ◽  
Number Of Binding Sites ◽  
Von Willebrand ◽  
Willebrand Factor

The effect of ristocetin on the binding of 125I-porcine von Willebrand factor to human platelets was studied. Previously, we had shown that 125I-porcine von Willebrand factor binds to human platelets in the absence of ristocetin. The present work demonstrates that binding is stimulated by ristocetin and this stimulation is maximal at a ristocetin concentration of 2 mg/ml. At a ristocetin concentration of 0.5 mg/ml, Scatchard analysis indicates a binding constant of 5.18 × 10-9M and the presence of 105,000 binding sites. This compares with our previous finding, in the absence of ristocetin, of a binding constant of 2.92 × 10-7M and 4760 binding sites. These binding data assume the porcine von Willebrand factor to be a tetramer with a molecular weight of 9 × 105. This study indicates that ristocetin causes tighter binding and increases the number of binding sites on human platelets for porcine von Willebrand factor. Unlabelled porcine von Willebrand factor competitively inhibits the specific binding of the labelled protein and gives a binding constant of 0.17 × 10-9M. Similar results were obtained using human von Willebrand factor.

Interaction of Ca++ binding sites of troponin: significance for Ca++ movements

1975 ◽  
Vol 228 (1) ◽  
pp. 172-178 ◽  
Author(s):  
CR Honig ◽  
YS Reddy
Keyword(s):  
Muscle Contraction ◽  
Least Squares ◽  
Binding Sites ◽  
Time Course ◽  
Nonlinear Least Squares ◽  
Cooperative Effect ◽  
Active State ◽  
Binding Parameters ◽  
Binding Data ◽  
Intracellular Ca

Ca++ binding data for seven preparations of troponin and 37 preparations of native tropomyosin were analyzed by the method of nonlinear least squares. The analysis was based on the assumption that two classes of independent binding sites exist. Data from one-third the preparations could not be fitted with all binding parameters at true least-squares minimum, and computed values of parameters for two-thirds the preparations were biologically uniterpretable. We conclude that troponin does not contain two classes of independent binding sites. Comparison of Scratchard plots of Ca++ binding by troponin and native tropomyosin modifies binding through a cooperative effect on troponin. Certain features of the Scratchard plots are imcompatible with the possibility that troponin possess more than two classes of independent sites. We interpret these results to mean that troponin's binding sites interact and that the interaction is increased by tropomyosin.The interaction would cause Ca++ affinity to vary with time during a muscle contraction. The effect of variable Ca++ affinity on intracellular Ca++ movements and the time course of the active state is discussed.

scholarly journals Response of mouse proximal straight tubule and medullary thick ascending limb to beta-agonist.

1993 ◽  
Vol 4 (5) ◽  
pp. 1151-1158
Author(s):  
N S Morgunov ◽  
Y D You ◽  
D J Hirsch
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Beta Agonist ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Beta Receptor ◽  
Medullary Thick Ascending Limb ◽  
Binding Data ◽  
Straight Tubules

Scatchard analysis of (3H)CGP-12177 and (125I)cyanopindolol (CYP) radioligand binding data revealed the presence of specific beta-adrenergic receptor-binding sites on microdissected mouse proximal straight and medullary thick ascending limb (mTAL) tubules. beta-Receptor (10(-6) M isoproterenol) stimulation of isolated perfused mTAL tubules produced a consistent hyperpolarization of the transepithelial potential that was blocked by propranolol (10(-6) M), a beta-receptor antagonist, and furosemide (10(-4) M), an inhibitor of the Na+/K+/2 Cl- triporter. In contrast, there was no electrogenic response to isoproterenol stimulation in isolated perfused proximal straight tubules. In summary, radioligand binding data show that both proximal straight and mTAL tubules possess beta-receptor-binding sites. Electrogenic transport in the mTAL can be modulated by beta-agonists, but there was no detectable electrogenic response to beta-receptor stimulation in proximal straight tubules.

Ontogeny of ACTH(1–24) receptors in rat adrenal glands during the perinatal period

1989 ◽  
Vol 123 (3) ◽  
pp. 421-428 ◽  
Author(s):  
A. Chatelain ◽  
P. Durand ◽  
E. Naaman ◽  
J. P. Dupouy
Keyword(s):  
Binding Sites ◽  
Adrenal Glands ◽  
Perinatal Period ◽  
Scatchard Analysis ◽  
Newborn Rats ◽  
Excellent Correlation ◽  
Temperature Dependent ◽  
Single Class ◽  
Binding Data ◽  
Cytoplasmic Structures

ABSTRACT Binding of ACTH to receptors was studied on crude adrenal membranes from fetal and newborn rats. 125I-Labelled ACTH(1–24) was used as the radioligand, the steroidogenic potency of which was 100-fold lower than that of unlabelled ACTH(1–24). Binding was specific, rapidly equilibrated and temperature dependent. Scatchard analysis of the binding data revealed a single class of binding sites with a dissociation constant of about 100 nmol/l at all stages of development studied. The concentration of ACTH receptors expressed per mg membrane proteins decreased in fetuses between days 17 and 21 of gestation and remained stable in newborn rats from weeks 1 to 4. The number of ACTH receptors expressed per adrenal increased regularly in fetal and newborn rats. The perinatal evolution of these concentrations of ACTH receptors is related to the increase in the size of the adrenals and the changes in cytoplasmic structures of the adrenocortical cells. When the number of ACTH-binding sites was expressed per μg DNA, maximum values occurred in fetuses on day 19 of gestation, and minimum values in newborn rats, 1 week after birth. There was an excellent correlation between the plasma levels of immunoreactive ACTH and corticosterone and the number of ACTH receptors per μg DNA during the perinatal period. Other results suggest that ACTH is able to up-regulate the number of its own receptors. Journal of Endocrinology (1989) 123, 421–428

Chronic and endogenous regulation of insulin receptors by catecholamines in adipocytes from patients with a phaeochromocytoma

1990 ◽  
Vol 10 (2) ◽  
pp. 201-207 ◽  
Author(s):  
M. C. Carranza ◽  
M. A. Simón ◽  
A. Torres ◽  
C. Calle
Keyword(s):  
Adipose Tissue ◽  
Binding Sites ◽  
Insulin Receptors ◽  
Human Adipose Tissue ◽  
Insulin Binding ◽  
Scatchard Analysis ◽  
Cooperative Model ◽  
Receptor Affinity ◽  
Site Model ◽  
Binding Data

Insulin binding in adipocytes from patients with a phaeochromocytoma (PH) approached that of the controls (C) at low and higher concentrations of unlabeled insulin. The apparent receptor affinity was unchanged (ED50: PH 0.50×10−9M and C0.60×10−9M). Scatchard analysis of the binding data using the negative cooperative model revealed a 46% decrease in the total number of receptors together with no changes in both K−e (PH 0.55×109M−1 and C 0.36×109M−1) and K−f (PH 0.13×109 M−1 and C 0.07×109 M−1). According to the two site model, an altered proportion in the two classes of insulin binding sites was detected. This was accompanied by a catecholamine-desensitization of the adipocytes to the antilipolytic action of insulin. These events could represent a final situation of a chronic and endogeneous regulation by high levels of catecholamines of insulin receptors in human adipose tissue.

scholarly journals Exposure of platelet fibrinogen-binding sites by collagen, arachidonic acid, and ADP: inhibition by a monoclonal antibody to the glycoprotein IIb-IIIa complex

Blood ◽  
1983 ◽  
Vol 61 (1) ◽  
pp. 140-148
Author(s):  
G Di Minno ◽  
P Thiagarajan ◽  
B Perussia ◽  
J Martinez ◽  
S Shapiro ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Arachidonic Acid ◽  
Binding Sites ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Scatchard Analysis ◽  
Single Class ◽  
Fibrinogen Binding ◽  
Binding Data ◽  
High Affinity Site

Following stimulation with adenosine diphosphate (ADP), collagen, or arachidonic acid, unstirred human platelet suspensions bind 125I- fibrinogen in a reaction that reaches completion within 30 min. Scatchard analysis of these binding data reveals two sets of binding sites with all 3 agents: a high affinity site (Kd 0.029–0.045 microM) binding 1000–1600 fibrinogen molecules per platelet, and a lower affinity site (Kd 1.2–2.0 microM) binding 46,000–76,000 fibrinogen molecules per platelet. At a concentration of apyrase that inhibited ADP-induced fibrinogen binding by greater than 85%, fibrinogen binding induced by collagen and arachidonic acid was only partially affected. This suggests that fibrinogen binding induced by collagen or arachidonic acid does not require released ADP. We isolated a monoclonal antibody, B59.2, which precipitated the glycoprotein IIb- IIIa complex from solubilized platelet membranes. Binding of labeled antibody to platelets before or after exposure to ADP, collagen, or arachidonic acid showed a single class of approximately 22,000 binding sites with Kd 0.019 microM. Binding of B59.2 was complete within 1 min and was not inhibited by EDTA. Preincubation of platelet suspensions with a 2.1 microM concentration of B59.2 caused inhibition of secretion and aggregation, but not of thromboxane-B2 synthesis, in response to 1 microgram/ml collagen, 40 microM arachidonic acid, or 4 microM ADP, concentrations of aggregating agents that produced complete aggregation and secretion in the absence of B59.2. At this concentration of B59.2, fibrinogen binding to stimulated platelets was inhibited by approximately 45%-55%. These data demonstrate that collagen and arachidonic acid can expose fibrinogen binding sites independently of released ADP; and that the glycoprotein IIb-IIIa complex is involved in secretion, aggregation, and fibrinogen binding, but not in thromboxane synthesis occurring in response to collagen, arachidonic acid, or ADP.

scholarly journals Cytochalasin B as a probe for the two hexose-transport systems in rat L6 myoblasts

1988 ◽  
Vol 251 (1) ◽  
pp. 63-72 ◽  
Author(s):  
S R Chen ◽  
T C Y Lo
Keyword(s):  
Plasma Membrane ◽  
Transport System ◽  
Binding Sites ◽  
Cytochalasin B ◽  
Transport Systems ◽  
Scatchard Analysis ◽  
Hexose Transport ◽  
Whole Cells ◽  
High Affinity ◽  
Binding Data

We have recently demonstrated that two hexose-transport systems are present in undifferentiated rat L6 myoblasts: D-glucose and 2-deoxy-D-glucose are preferentially transported by the high-affinity system, whereas 3-O-methyl-D-glucose is transported primarily by the low-affinity system. Mutant D23 is found to be defective only in the high-affinity hexose-transport system. The low-affinity transport system is much more sensitive to inhibition by cytochalasin B (CB). The present study examines the identity, properties and regulation of the CB-binding sites by measuring CB binding to both whole cells and plasma membrane. Scatchard analysis of the binding data revealed the presence of two CB-binding sites, namely CBH and CBL. These two sites differ not only in their affinity for CB, but their levels can also be differentially altered by various biochemical, physiological and genetic manipulations. CBL resembles the high-affinity hexose-transport system in that it is absent in mutant D23 and is present in larger quantities in glucose-starved cells. Moreover, CB binding to this site is inhibited by D-glucose and 2-deoxy-D-glucose, the preferred substrates of the high-affinity hexose-transport system. On the other hand, CBH is found to be unaltered in mutant D23, which also retains the normal low-affinity hexose-transport system. CBH also resembles the low-affinity transport system in that it is not elevated in glucose-starved cells. Furthermore, binding of CB to this site can be inhibited by 3-O-methyl-D-glucose, the preferred substrate of the low-affinity transport system. It should be noted that 2-deoxy-D-glucose does not have much effect on CBH, and vice versa. Studies with purified membrane preparations indicate that both CB-binding sites are present in similar ratios in the plasma membrane and the low-density microsomal fraction. Plasma-membrane studies also reveal that D-glucose 6-phosphate, but not 2-deoxy-D-glucose 6-phosphate, is very effective in activating CB binding. Data presented suggest that CB binding may be regulated by sugar analogues in an allosteric manner.

scholarly journals The structure of binding curves and practical identifiability of equilibrium ligand-binding parameters

2016 ◽  
Vol 149 (1) ◽  
pp. 121-147 ◽  
Author(s):  
Thomas R. Middendorf ◽  
Richard W. Aldrich
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Association Constants ◽  
Binding Parameters ◽  
Parameter Identifiability ◽  
Binding Parameter ◽  
Binding Data ◽  
Binding Curve ◽  
Site Binding ◽  
Binding Curves

A critical but often overlooked question in the study of ligands binding to proteins is whether the parameters obtained from analyzing binding data are practically identifiable (PI), i.e., whether the estimates obtained from fitting models to noisy data are accurate and unique. Here we report a general approach to assess and understand binding parameter identifiability, which provides a toolkit to assist experimentalists in the design of binding studies and in the analysis of binding data. The partial fraction (PF) expansion technique is used to decompose binding curves for proteins with n ligand-binding sites exactly and uniquely into n components, each of which has the form of a one-site binding curve. The association constants of the PF component curves, being the roots of an n-th order polynomial, may be real or complex. We demonstrate a fundamental connection between binding parameter identifiability and the nature of these one-site association constants: all binding parameters are identifiable if the constants are all real and distinct; otherwise, at least some of the parameters are not identifiable. The theory is used to construct identifiability maps from which the practical identifiability of binding parameters for any two-, three-, or four-site binding curve can be assessed. Instructions for extending the method to generate identifiability maps for proteins with more than four binding sites are also given. Further analysis of the identifiability maps leads to the simple rule that the maximum number of structurally identifiable binding parameters (shown in the previous paper to be equal to n) will also be PI only if the binding curve line shape contains n resolved components.

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