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.

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.

Increased beta-adrenergic receptors in brown fat of winter-acclimatized Alaskan voles

1983 ◽  
Vol 245 (3) ◽  
pp. R357-R363 ◽  
Author(s):  
D. D. Feist
Keyword(s):  
Cold Acclimation ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Specific Binding ◽  
Brown Fat ◽  
Scatchard Analysis ◽  
Beta Adrenergic Receptors ◽  
Hormonal Stimulation ◽  
Beta Adrenergic ◽  
Specific Binding Sites

To assess a possible mechanism for the enhanced thermogenesis of cold-acclimated and winter-acclimatized red-backed voles (Clethrionomys rutilus), beta-adrenergic receptors of brown fat were characterized by specific binding of (-)-[3H]-dihydroalprenolol [( 3H]DHA) to isolated brown fat membranes from 23 degrees C-acclimated controls, cold-acclimated (5 wk or 5 mo at 5 degrees C), wild summer, and winter-acclimatized voles. Scatchard analysis to determine the equilibrium dissociation constant (Kd) and the maximum number of binding sites (Bmax) for control brown fat membranes gave a Kd of 4.45 nM [3H]DHA and Bmax of 249 fmol [3H]DHA bound per milligram of protein. beta-Adrenergic agonists competed for specific binding sites with an order of potency typical of the beta 1 subtype of adrenergic receptors: (-)-isoproterenol greater than (-)-norepinephrine greater than or equal to (-)-epinephrine. After cold acclimation for 5 wk or 5 mo, the Kd and Bmax for adrenergic binding sites were similar to those of controls. Brown fat mass was 1.5 times greater than that of controls after 5 wk cold acclimation but similar to controls after 5 mo cold acclimation. Winter voles had 1.7 times higher Bmax and 1.6 times more brown fat than summer voles. Thus seasonal acclimatization to winter in red-backed voles appears to involve an increase in beta-adrenergic receptors in brown fat, but cold acclimation does not. The results suggest quantitative and possibly qualitative differences in neural and hormonal stimulation of brown fat between cold acclimation and winter acclimatization in voles.

scholarly journals Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland

1998 ◽  
Vol 157 (1) ◽  
pp. 99-106 ◽  
Author(s):  
G Muccioli ◽  
C Ghe ◽  
MC Ghigo ◽  
M Papotti ◽  
E Arvat ◽  
...  
Keyword(s):  
Substance P ◽  
Human Brain ◽  
Pituitary Gland ◽  
Binding Sites ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Gh Secretagogues ◽  
Specific Binding Sites ◽  
Specific Receptors

In vitro studies have been performed to demonstrate and characterize specific binding sites for synthetic GH secretagogues (sGHS) on membranes from pituitary gland and different human brain regions. A binding assay for sGHS was established using a peptidyl sGHS (Tyr-Ala-hexarelin) which had been radioiodinated to high specific activity at the Tyr residue. Specific binding sites for 125I-labelled Tyr-Ala-hexarelin were detected mainly in membranes isolated from pituitary gland and hypothalamus, but they were also present in other brain areas such as choroid plexus, cerebral cortex, hippocampus and medulla oblongata with no sex-related differences. In contrast, negligible binding was found in the thalamus, striatum, substantia nigra, cerebellum and corpus callosum. The binding of 125I-labelled Tyr-Ala-hexarelin to membrane-binding sites is a saturable and reversible process, depending on incubation time and pH of the buffer. Scatchard analysis of the binding revealed a finite number of binding sites in the hypothalamus and pituitary gland with a dissociation constant (Kd) of (1.5 +/- 0.3) x 10(-9) and (2.1 +/- 0.4) x 10(-9) mol/l respectively. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipids are essential for the binding of 125I-labelled Tyr-Ala-hexarelin. The binding of 125I-labelled Tyr-Ala-hexarelin to pituitary and hypothalamic membranes was displaced in a dose-dependent manner by different unlabelled synthetic peptidyl (Tyr-Ala-hexarelin, GHRP2, hexarelin, GHRP6) and non-peptidyl (MK 0677) sGHS. An inhibition of the specific binding was also observed when binding was performed in the presence of [D-Arg1-D-Phe5-D-Trp7,9-Leu11]-substance P, a substance P antagonist that has been found to inhibit GH release in response to sGHS. In contrast, no competition was observed in the presence of other neuropeptides (GHRH, somatostatin, galanin or Met-enkephalin) which have a known influence on GH release. In conclusion, the present data demonstrate that sGHS have specific receptors in human brain and pituitary gland and reinforce the hypothesis that these compounds could be the synthetic counterpart of an endogenous GH secretagogue involved in the neuroendocrine control of GH secretion and possibly in other central activities.

scholarly journals Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver

1984 ◽  
Vol 219 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
Y A Lefebvre ◽  
J T Venkatraman
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Intact Animal ◽  
Binding Capacity ◽  
Specific Binding ◽  
Male Rat ◽  
Scatchard Analysis ◽  
Specific Binding Sites ◽  
Number Of Binding Sites

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3′,5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3′,5′-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.

HIGH-AFFINITY BINDING OF REVERSE T3 AND TETRAC IN NUCLEI OF HUMAN LYMPHOCYTES

1978 ◽  
Vol 87 (3) ◽  
pp. 516-524 ◽  
Author(s):  
T. Lemarchand-Béraud ◽  
A.-C. Holm ◽  
G. Bornand ◽  
A. Burger
Keyword(s):  
Human Lymphocyte ◽  
Binding Sites ◽  
Specific Binding ◽  
Human Lymphocytes ◽  
Affinity Constant ◽  
Reverse T3 ◽  
Intact Cells ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Direct Measurements

ABSTRACT In a previous study, human lymphocyte nuclei were found to possess high affinity, low capacity binding sites for triiodothyronine (T3) and thyroxine (T4). The number of receptors per cell was similar for T3 and T4 (115±20), but the equilibrium affinity constant (Ka) for T3 (2.20±0.23 1010m−1) was twice that for T4 (1.05 ± 0.25 1010m−1). The present study shows that human lymphocyte nuclei also bind highly purified [125I]tetrac and [125I]rT3. The number of specific binding sites was 60 for tetrac and 40 for rT3. The Ka for tetrac (2.12 ± 0.29 1010m−1) was similar to that of T3, whereas that of rT3 (1.31 ± 0.2110m−1) was similar to that of T4. The Ka was the same when measured in intact cells and in nuclei isolated after incubation. Despite the similar Ka for tetrac, rT3 and T3, as obtained by direct measurements, tetrac had only 2 % and rT3 0.1 % of the T3 potency in T3 displacement studies. [125I] tetrac was displaced 50% by 20 fmol of T3 and [125I]rT3 by 8 fmol. These results show that tetrac and rT3 do bind as strongly to nuclear receptors as T3 and T4, but that when competing with T3 the apparent affinities decrease considerably for tetrac and rT3. Thus, the nuclear binding of these two analogues probably has no significance under physiological conditions, but may play some role under pathological conditions when the formation of T3 is decreased and that of rT3 and tetrac is increased. This may represent an adaptive mechanism in T4 inactivation.

Modulation of leukotriene B4 and platelet-activating factor binding to neutrophils

1991 ◽  
Vol 261 (3) ◽  
pp. C515-C520 ◽  
Author(s):  
M. Yamazaki ◽  
T. F. Molski ◽  
T. Stevens ◽  
C. K. Huang ◽  
E. L. Becker ◽  
...  
Keyword(s):  
Pertussis Toxin ◽  
Specific Binding ◽  
Platelet Activating Factor ◽  
Leukotriene B4 ◽  
Arachidonic Acid Metabolism ◽  
Human Neutrophils ◽  
Lipoxygenase Pathway ◽  
Intact Cells ◽  
Gm Csf ◽  
Protein Kinase C Inhibitor

Preincubation of human neutrophils with the human hormone granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibits the specific binding of leukotriene B4 ([3H]LTB4) but not the nonmetabolizable bioactive platelet-activating factor ([3H]C-PAF) to intact cells. This inhibition requires that the GM-CSF interacts with intact cells. The action of GM-CSF is not prevented by pertussis toxin. Moreover, the rise in calcium produced by LTB4 but not by PAF is also inhibited in human neutrophils pretreated with GM-CSF. Interestingly, neither the inhibitory action of GM-CSF on [3H]LTB4 binding or LTB4-induced calcium rise nor the potentiation of superoxide production by GM-CSF is reduced by inhibitors of arachidonic acid metabolism by the lipoxygenase pathway. In contrast, preincubation of human neutrophils with either the chemotactic factor formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) or the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), inhibits the binding of both [3H]LTB4 and [3H]C-PAF to intact cells. The inhibitory actions of GM-CSF, PMA, and fMet-Leu-Phe require that they interact with the intact cells; their actions cannot be reproduced in plasma membrane preparations. The effects of both GM-CSF and fMet-Leu-Phe cannot be prevented by the protein kinase C inhibitor staurosporine. The mechanisms of fMet-Leu-Phe and GM-CSF actions are probably not mediated through the release of LTB4 by the cells. Interestingly, this new action, unlike other reported effects of GM-CSF, is not mediated through a pertussis toxin-sensitive G protein (Gi alpha 2). This indicates that not all GM-CSF receptors are coupled to Gi alpha 2.

scholarly journals Inositol 1,3,4,5-tetrakisphosphate binding sites in neuronal and non-neuronal tissues. Properties, comparisons and potential physiological significance

1992 ◽  
Vol 288 (1) ◽  
pp. 149-154 ◽  
Author(s):  
P J Cullen ◽  
R F Irvine
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
High Specificity ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Intact Cells ◽  
Peripheral Tissues ◽  
Ligand Displacement ◽  
Neuronal Tissues ◽  
A Site

1. Ins(1,3,4,5)P4 binding sites were studied in cerebellar and hepatic microsomes from rat, and in bovine adrenal-cortical microsomes. 2. At pH 7.0, all three tissues showed specific binding, with Ins(1,3,4,5)P4 being the most potent competing ligand of those tested [which included Ins(1,4,5)P3, Ins(1,3,4,5,6)P5 and InsP6] and Scatchard analysis suggested two sites; a site with high affinity and high specificity [Kd (1-6) x 10(-9) M] and a site with low affinity and low specificity [Kd (2-6) x 10(-7) M]. 3. At pH 5.5, cerebellar and bovine adrenal microsomes showed similar binding properties: two affinities with a similar specificity for Ins(1,3,4,5)P4 as at pH 7.0. 4. However, when assayed in a low-ionic strength acetate-based buffer at pH 5.0, cerebellar microsomes retain specific Ins(1,3,4,5)P4 binding sites, whereas bovine adrenal and hepatic microsomal binding sites lose much of their specificity, as InsP6 and Ins(1,3,4,5,6)P5 are equally as potent as Ins(1,3,4,5)P4. 5. Pi (25 mM), which is frequently included in Ins(1,3,4,5)P4 binding assays, had a small inhibitory effect on binding of cerebellar and adrenal microsomes at pH 5.5, but a large effect at pH 7.0, so that a considerable decrease occurs in the amount of specific binding at pH 5.5 compared with that at pH 7.0, if Pi is omitted from the binding assay. 6. Cerebellar and adrenal microsomes were used in a ligand-displacement mass assay (conducted under near-physiological conditions, at pH 7.0) on extracts of cerebral-cortex slices stimulated with agonists, and both preparations faithfully detected the increases in Ins(1,3,4,5)P4 that occurred, implying that Ins(1,3,4,5)P4 is the principal ligand on these binding sites in intact cells. 7. Apparent contradictions in the literature with regard to Ins(1,3,4,5)P4 binding sites in neuronal and peripheral tissues can be largely accounted for by the data, and the properties of the binding sites detected at physiological pH are consistent with the possibility that they are putative receptors for the proposed second-messenger role for Ins(1,3,4,5)P4.

scholarly journals Membrane receptor for odour-binding proteins

1996 ◽  
Vol 317 (1) ◽  
pp. 23-27 ◽  
Author(s):  
Mohamed BOUDJELAL ◽  
Asipu SIVAPRASADARAO ◽  
John B. C FINDLAY
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Binding Protein ◽  
Structural Similarity ◽  
Thiol Group ◽  
The Body ◽  
Retinol Binding Protein ◽  
Proteinase K ◽  
Scatchard Analysis ◽  
Major Urinary Proteins

Specific binding of 125I-labelled bovine odour-binding protein (OBP) to isolated membranes from nasal mucosa was demonstrated. The interaction reached equilibrium within 30 min at 37 °C and was reversible. A Scatchard analysis of the equilibrium binding revealed a single population of binding sites, with the calculated equilibrium dissociation constant and maximum number of binding sites being 2.25±0.5 μM and 18.5±2 pmol/mg of membrane protein respectively (n = 2). Receptor activity was decreased on digestion by trypsin, proteinase K or endoglycosidase H, was heat labile and was sensitive to thiol-group-specific reagents. With the exception of rat and mouse major urinary proteins, which exhibit a high degree of structural similarity with OBP and bind similar ligands, other members of the lipocalin family, such as retinol-binding protein and β-lactoglobulin, failed to inhibit the binding of 125I-labelled OBP to its receptor. The receptor seems not to be restricted to olfactory tissues, as it was detected in a variety of other tissues. This suggests that OBP is unlikely to play a role only in olfactory signal transduction. It might have a much broader role within the body; possibilities include a role in detoxification or signalling.

Calcium and the Fc Receptor on Human Platelets

1987 ◽  
Vol 57 (03) ◽  
pp. 298-301
Author(s):  
William F Clark ◽  
Gerald J M Tevaarwerk ◽  
Bruce D Reid ◽  
Suzanne Hall ◽  
Anita Caveney ◽  
...  
Keyword(s):  
Specific Binding ◽  
Normal Subjects ◽  
Human Platelets ◽  
Fc Receptor ◽  
Normal Male ◽  
Scatchard Analysis ◽  
Normal Female ◽  
Apparent Difference ◽  
Binding Data ◽  
Direct Binding

SummaryWe have described the calcium dependence of the IgG Fc receptor (Fc-R) on human platelets by analyzing the direct binding of radiolabelled Fc fragments, monomers and dimers of IgG. Specific binding to platelets was undetectable at 37° C in a calcium-free preparation but readily detected when calcium was restored. Scatchard analysis of the binding data for the calcium-restored platelets permitted calculation of the available Fc-R and the Ka of binding for the different IgG ligands. The mean Ka of binding for 12 normal subjects varied from 107 to 108 L/M, with an equal receptor number measured by Fc fragments and dimers of IgG, but a lesser amount for monomeric IgG. There was no apparent difference in Fc-R number for platelets from 6 normal male versus 6 normal female subjects.At 4° C binding was detectable for dimers and polymers of IgG in a calcium-free preparation and this was markedly increased with recalcification. Thus, our data are consistent with an Fc receptor population on human platelets whose avidity for binding is significantly enhanced in a calcium-restored medium.

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