scholarly journals Presence of sterol-binding sites in the cytosol of French-bean (Phaseolus vulgaris) roots

1988 ◽  
Vol 250 (2) ◽  
pp. 565-569 ◽  
Author(s):  
M A Vega ◽  
R L Boland
Keyword(s):  
Phaseolus Vulgaris ◽  
Vitamin D3 ◽  
Binding Sites ◽  
Specific Binding ◽  
French Bean ◽  
Binding Reaction ◽  
Equilibrium Binding ◽  
Tissue Responses ◽  
Equilibrium Binding Constant ◽  
Sterol Binding

Vitamin D3 (cholecalciferol) and stigmasterol have been shown to stimulate Ca2+ uptake and to induce calmodulin synthesis in cultured French-bean (Phaseolus vulgaris) roots. In addition, the appearance of calmodulin in the cultures in response to vitamin D3 could be prevented by RNA-synthesis inhibitors. To investigate the possibility that the sterols affect root DNA transcription through a receptor-mediated mechanism, the existence of sterol-binding sites in P. vulgaris roots was investigated. Specific binding of [3H]vitamin D3 could be demonstrated with intact tissue and the cytosolic fraction obtained therefrom. Equilibrium in the binding reaction with cytosol was attained after 4 h of incubation at 0 degrees C. The [3H]vitamin D3 was reversibly bound, since it could be displaced by an excess of unlabelled sterol. An equilibrium binding constant (KD) of (3.48 +/- 0.09) x 10(-9) M and a maximum binding-site concentration (nmax) of 32 +/- 2.54 (3) pmol/mg of protein could be calculated by Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672] analysis. In addition to vitamin D3, stigmasterol and sitosterol were effectively able to compete with [3H]vitamin D3 for binding to root cytosol. Cortisol, oestradiol and progesterone displaced bound labelled vitamin D3 to a lesser extent, whereas 5 beta-dihydrotestosterone, lanosterol and diosgenin were ineffective. The affinity and specificity of the root sterol-binding sites are in agreement with the characteristics of tissue responses to the sterols (Ca2+ uptake and calmodulin synthesis).

scholarly journals Mannose-specific binding sites for horseradish peroxidase in various cells of the rat.

1983 ◽  
Vol 31 (1) ◽  
pp. 78-84 ◽  
Author(s):  
W Straus
Keyword(s):  
Mast Cells ◽  
Horseradish Peroxidase ◽  
Binding Sites ◽  
Specific Binding ◽  
Cell Types ◽  
Sinusoidal Cells ◽  
Binding Reaction ◽  
Specific Binding Sites ◽  
Liver Sinusoidal Cells ◽  
Endogenous Lectins

Mannose-specific binding sites for horseradish peroxidase (HRP) were studied in fixed sections of various tissues by a method reported previously. Liver sinusoidal cells, mast cells of lymph nodes, and alveolar macrophages of the lung and skin fibroblasts were main cell types showing mannose-specific binding of HRP. Macrophages, fibroblasts, and mast cells in the connective tissue of other organs also showed the reaction. However, macrophages of the spleen, and cultured 3T3 cells and L-cells did not give the reaction. The specificities of the binding reaction were studied by determining the approximate concentrations of competing sugars that suppressed the specific binding of HRP. It was found that the endogenous lectins in macrophages, fibroblasts, mast cells, and liver sinusoidal cells showed similar specificities toward various carbohydrates. D-Mannose and L-fucose had the highest affinity toward the lectins (competing ability for the binding of HRP). D-Mannose-6-phosphate, N-acetyl-D-glucosamine, D-glucose, D-ribose, and D-arabinose showed intermediate affinity, whereas D-xylose and D-galactose showed low affinity. Polymerized mannose in mannan and glycoproteins rich in mannose groups (invertase and ribonuclease B) showed much higher affinity to the binding sites than free mannose.

scholarly journals The binding of ouabain to normal and chronic lymphocytic leukemic lymphocytes

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 994-1000
Author(s):  
JS Wiley ◽  
N Kraft ◽  
IA Cooper
Keyword(s):  
Peripheral Blood ◽  
Binding Sites ◽  
Specific Binding ◽  
Lymphocytic Leukemia ◽  
Ouabain Binding ◽  
Normal Peripheral Blood ◽  
Binding Characteristics ◽  
Specific Binding Sites ◽  
Equilibrium Binding ◽  
The Difference

The binding of the cardiac glycoside, ouabain, to cells had been used to quantify the number of active cation pumps. In this study, lymphocytes were incubated with 3H-ouabain and the equilibrium binding analyzed for the maximal number of specific binding sites. Lymphocytes from normal peripheral blood bound 44,200 +/- 9920 molecules/cell, compared with 29,200 +/- 8370 molecules/cell for the lymphocytes of chronic lymphocytic leukemia (CLL) subjects. This difference was significant (p less than 0.01) and did not reflect a lower number of sites on B cells than T cells, since B-cell-enriched lymphocytes from normal peripheral blood showed the same ouabain binding characteristics as the standard T-cell-rich preparation. Although monocytes bind threefold more ouabain than lymphocytes, the small monocyte contamination (3.0%) in normal lymphocyte preparations could not account for the difference between normal and CLL. The fewer ouabain binding sites on CLL lymphocytes may reflect both their smaller size (by 10%) and lower mitotic activity compared with lymphocytes from normal peripheral blood.

Quantitative determination of equilibrium binding isotherms for multiple ligand-macromolecule interactions using spectroscopic methods

2000 ◽  
Author(s):  
Wlodzimierz Bujalowski ◽  
Maria J. Jezewska
Keyword(s):  
Ligand Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
Equilibrium Constants ◽  
Free Ligand ◽  
Ligand Concentration ◽  
Equilibrium Binding ◽  
Theoretical Predictions ◽  
Molecular Forces ◽  
Molecular Aspects

Thermodynamic studies provide information that is necessary in order to understand the forces that drive the formation of ligand-macromolecule complexes. Knowledge of the energetics of these interactions is also indispensable for characterization of functionally important structural changes that occur within the studied complexes. Quantitative examination of the equilibrium interactions are designed to provide the answers to the questions: What is the stoichiometry of the formed complexes? How strong or how specific are the interactions? Are there any cooperative interactions among the binding sites and/or the bound ligand molecules? Are the binding sites intrinsically heterogeneous? What are the molecular forces involved in the formation of the studied complexes, or, in other words, how do the equilibrium binding and kinetic parameters depend on solution variables (temperature, pressure, pH, salt concentration, etc.)? Equilibrium isotherms for the binding of a ligand to a macromolecule represent the relationship between the degree of ligand binding (moles of ligands bound per mole of a macromolecule) and the free ligand concentration. A true thermodynamic binding isotherm is model-independent and reflects only this relationship. Only then, when such an isotherm is obtained, can one proceed to extract physically meaningful interaction parameters that characterize the free energies of interaction. This is accomplished by comparing the experimental isotherms to theoretical predictions based on specific binding models that incorporate known molecular aspects, such as intrinsic binding constants, cooperativity parameters, allosteric equilibrium constants, discrete character of the binding sites or overlap of potential binding sites, etc. (see below). Any method used to quantitatively study ligand binding to a macromolecule must relate the extent of the complex formation to the free ligand concentration in solution. Numerous techniques have been developed to study equilibrium properties of specific and non-specific ligand-macromolecule interactions in which binding is directly monitored, including equilibrium dialysis, ultrafiltration, column chromatography, filter binding assay and gel electrophoresis (1-6). These direct methods are very straightforward; however, they are usually time consuming and some, like filter binding or gel shift assays, are non-equilibrium techniques which require many controls before the reliable equilibrium binding data can be obtained.

scholarly journals The binding of ouabain to normal and chronic lymphocytic leukemic lymphocytes

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 994-1000 ◽  
Author(s):  
JS Wiley ◽  
N Kraft ◽  
IA Cooper
Keyword(s):  
Peripheral Blood ◽  
Binding Sites ◽  
Specific Binding ◽  
Lymphocytic Leukemia ◽  
Ouabain Binding ◽  
Normal Peripheral Blood ◽  
Binding Characteristics ◽  
Specific Binding Sites ◽  
Equilibrium Binding ◽  
The Difference

Abstract The binding of the cardiac glycoside, ouabain, to cells had been used to quantify the number of active cation pumps. In this study, lymphocytes were incubated with 3H-ouabain and the equilibrium binding analyzed for the maximal number of specific binding sites. Lymphocytes from normal peripheral blood bound 44,200 +/- 9920 molecules/cell, compared with 29,200 +/- 8370 molecules/cell for the lymphocytes of chronic lymphocytic leukemia (CLL) subjects. This difference was significant (p less than 0.01) and did not reflect a lower number of sites on B cells than T cells, since B-cell-enriched lymphocytes from normal peripheral blood showed the same ouabain binding characteristics as the standard T-cell-rich preparation. Although monocytes bind threefold more ouabain than lymphocytes, the small monocyte contamination (3.0%) in normal lymphocyte preparations could not account for the difference between normal and CLL. The fewer ouabain binding sites on CLL lymphocytes may reflect both their smaller size (by 10%) and lower mitotic activity compared with lymphocytes from normal peripheral blood.

Study on the Disaggregation of Photosensitizer Pentalysine β-Carbonyl-Phthalocyanine Zinc In Vitro

2011 ◽  
Vol 335-336 ◽  
pp. 363-367
Author(s):  
Chun Yan Li ◽  
Guan Yu Ruan ◽  
Sheng Xiong Dong ◽  
Xiao Li Shi ◽  
Jin Can Chen ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Photodynamic Therapy ◽  
Human Serum Albumin ◽  
Molecular Interaction ◽  
Binding Sites ◽  
Molecular Model ◽  
Equilibrium Binding ◽  
Equilibrium Binding Constant

A novel photosensitizer, pentalysine β-carbonyl-phthalocyanine zinc [ZnPc-(Lys)5] has tendency to form aggregate in aqueous solution. The observed in vivo Photodynamic therapy (PDT) effect of ZnPc-(Lys)5suggests a disaggregation mechanism. In this study, the equilibrium binding constant Ka, the numbers of binding sites n and the distance of Forster radii r between ZnPc-(Lys)5and human serum albumin (HSA) are measured by Spectroscopy. A molecular model of HSA-ZnPc-(Lys)5complex was generated according to these datum. This molecular model provides rationale that the molecular interaction between HSA and ZnPc-(Lys)5facilitates the dissociation of ZnPc aggregates.

[125I]Iodomelatonin-binding sites in the bursa of Fabricius of birds: binding characteristics, subcellular distribution, diurnal variations and age studies

1993 ◽  
Vol 138 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Z. M. Liu ◽  
S. F. Pang
Keyword(s):  
Binding Sites ◽  
Subcellular Distribution ◽  
Specific Binding ◽  
Binding Constants ◽  
Bursa Of Fabricius ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
Equilibrium Binding ◽  
Number Of Binding Sites ◽  
Age Studies

ABSTRACT The melatonin-binding sites in membrane preparations of the bursa of Fabricius of birds were studied using [125I]iodomelatonin as a radioligand. The binding sites were stable, saturable, reversible and of high affinity. Scatchard analysis of specific binding revealed equilibrium binding constants (Kd) of (means±s.e.m.) 43·1 ±5·9 73·3±7·6 and 35·3±4·8 pmol/l respectively at the mid-point of the light period (mid-light) in chickens, pigeons and quail, with a total number of binding sites (Bmax) of 1·23 ±0·15, 1·33±0·18 and 0·94 ±0·07 fmol/mg protein. The diurnal variation in [125I]iodomelatonin binding showed that the Bmax was 45, 115 and 70% higher at mid-light than at mid-dark in the bursae of chickens, pigeons and quail respectively. The Kd value determined from kinetic analysis was 49·0 ±6·4 pmol/l at mid-light in the chicken bursa. The [125I]iodomelatonin-binding sites of chicken bursal membranes had the following order of pharmacological affinities: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin > 5-hydroxytryptamine, tryptamine, 5-methoxytryptophol, 1-acetylindole-3-carboxaldehyde, 3-acetylindole, l-tryptophan, 5-hydroxyindole3-acetic acid, 5-hydroxytryptophan suggesting that the [125I]iodomelatonin-binding sites were highly specific for melatonin. The subcellular distribution of binding sites in the chicken bursa was in the following descending order: nuclear > mitochondrial > microsomal > cytosolic fraction. There was an agerelated decrease in [125I]iodomelatonin-binding in chicken bursal membranes, with higher densities in the neonate. Our studies of [125I]iodomelatonin-binding sites in the bird bursa indicate that this primary lymphoid tissue is a target organ for melatonin, and that melatonin has a direct effect on the immune system. Journal of Endocrinology (1993) 138, 51–57

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

Deficiency of (33P)2MeS-ADP Binding Sites on Platelets with Secretion Defect, Normal Granule Stores and Normal Thromboxane A2 Production

1997 ◽  
Vol 77 (05) ◽  
pp. 0986-0990 ◽  
Author(s):  
Marco Cattaneo ◽  
Rossana Lombardi ◽  
Maddalena L Zighetti ◽  
Christian Gachet ◽  
Philippe Ohlmann ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Thromboxane A2 ◽  
Normal Subjects ◽  
Congenital Defects ◽  
Normal Production ◽  
Adp Receptor ◽  
Partial Deficiency ◽  
Induced Aggregation ◽  
Platelet Secretion

SummaryBy the term “Primary Secretion Defect” (PSD), we mean a common heterogeneous group of congenital defects of platelet secretion, characterized by a normal primary wave of platelet aggregation induced by ADP and other agonists, a normal concentration of platelet granule contents, and normal production of thromboxane A2. The biochemical abnormalities responsible for PSD are not well known. Since a secretion defect similar to PSD is found in platelets that are severely deficient of binding sites for the ADP analogue 2MeS-ADP and do not aggregate in response to ADP, we tested the hypothesis that PSD platelets have moderately decreased 2MeS-ADP binding sites, which may be sufficient for normal ADP-induced aggregation but not for potentiating platelet secretion. The specific binding of [33P]2MeS-ADP to platelets from 3 PSD patients (347,443 and 490 sites/platelet; KD 2.8-3.9 nM) was lower than to platelets from 24 normal subjects (647 [530-1102]; KD = 3.8 [2.3-7.3]) (median [range]). Normal values were found in a fourth PSD patient (710; KD 3.7). The degree of inhibition of PGE1- induced cAMP increase by 0.1 |μM ADP was lower in patients than in controls. The secretion induced by the endoperoxide analogue U46619 from normal, acetylsalicylic acid-treated platelets under conditions that prevented the formation of large aggregates was potentiated by 1 fimol/1 ADP and inhibited by apyrase. These findings indicate that a partial deficiency of the platelet ADP receptor(s) might be responsible for the defect of platelet secretion in some PSD patients and that ADP potentiates platelet secretion independently of the formation of large aggregates and thromboxane A2 production.

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