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.

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 Binding of Calcium to Fibrinogen: Some Related Properties

1977 ◽  
Author(s):  
G. Marguerie
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Specific Binding ◽  
Equilibrium Dialysis ◽  
Structural Features ◽  
Salt Bridges ◽  
High Affinity ◽  
Direct Titration ◽  
Specific Binding Sites ◽  
Calcium Binding Sites

The calcium binding properties of bovin fibrinogen have been studied using equilibrium dialysis method. At pH 7.5 fibrinogen has 3 specific calcium binding sites of high affinity and several non specific binding sites of low affinity. Direct titration of the calcium induced proton release indicates that the binding center is a chelate. Thermal an acid denaturation is found to be markedly influenced by the presence of Ca++, suggesting that structural features are related to the binding. However the circular dichroism spectra show that no generalized conformational change is induced when Ca++ is bound to the protein.The plasminic digestion of fibrinogen is also found to be specificaly influenced by Ca++. The velocity of the initial cleavages is slightly reduced in the presence of calcium. It is therefore suggested that the C-terminal part of the Aα chain is involved in the binding.Considering the dimeric structure of the fibrinogen molecule, the presence of only 3 calcium binding sites of high affinity suggests the existence of “salt bridges” between the constitutive polypeptide chains.

EFFECT OF Ca2+ and Mg2+ ON PLATELET ACTIVATING FACTOR (PAF) INDUCED AGGREGATION AND SPECIFIC BINDING TO HUMAN PLATELETS

1987 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer
Keyword(s):  
Platelet Aggregation ◽  
Calcium Channel ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Specific Binding ◽  
Human Platelets ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Significant Difference ◽  
Induced Aggregation

Gel filtered human platelets (GFP) collected in Tyrode's buffer containing 0.5 mM Ca+2, ImM Mg+2, and 0.35% albumin exhibit high affinity binding of 3H-PAF with a Kd of 0.109 α 0.029 nM (mean α SD; n=13) and 267 α 70 sites per platelet. When fibrinogen (1.67 mg/ml final concentration) is added to these GFP preparations biphasic aggregation is observed with PAF (4 nM). Normal aggregation is also observed with other platelet agonists including ADP, epinephrine, collagen, arachidonic acid, A23187 and thrombin. If GFP is prepared without added Ca+2 or Mg+2 in the presence of 3mM EDTA, platelets do not aggregate in response to PAF. However the number of specific binding sites remains unchanged (387 per platelet) with some decrease in affinity of binding (Kd = 0.2l4nM). In the presence of ImM Mg+2 there is no significant difference in binding kinetics over a range of Ca+2 concentrations (0-2mM). On the other hand the calcium channel blocker verapamil (5-10uM) exhibits competitive inhibition of 3H-PAF as analyzed by Lineweaver-Burk plots. Specific binding of 3H-PAF to GFP in the presence of ImM Mg+2 and ImM EGTA shows Kd of 0.l66nM but with increase in specific binding sites to 665. Despite increase in number of sites and no change in binding affinity, GFP under these conditions does not exhibit platelet aggregation with PAF in doses up to 80 nM.From these data it appears that external Ca+2 is not necessary for specific binding of 3H-PAF to its high affinity receptor. However, calcium does appear to be necessary for second wave aggregation with PAF. While Mg+2 appears to enhance 3H-PAF binding to platelets Mg+2 cannot substitute for Ca+2 in PAF induced platelet aggregation. Although verapamil appears to competitively inhibit binding of PAF to GFP it is not clear whether the inhibition is due to competition at or near the actual PAF receptor or at a site involving the calcium channel.

TRIIODOTHYRONINE AND THYROXINE NUCLEAR RECEPTORS IN LYMPHOCYTES FROM NORMAL, HYPER- AND HYPOTHYROID SUBJECTS

1977 ◽  
Vol 85 (1) ◽  
pp. 44-54 ◽  
Author(s):  
Th. Lemarchand-Béraud ◽  
A.-Ch. Holm ◽  
B. R. Scazziga
Keyword(s):  
Nuclear Receptors ◽  
Binding Sites ◽  
Intact Cell ◽  
Affinity Constant ◽  
Limited Capacity ◽  
Intact Cells ◽  
High Affinity ◽  
The Mean ◽  
Hypothyroid Patients ◽  
Hyperthyroid Patients

ABSTRACT In an investigation of thyroxine (T4) and triiodothyronine (T3) receptors in humans, the lymphocyte was chosen as the target cell. This study was performed to elucidate whether T3 and T4 bind to different receptors, if T4 is bound only after conversion into T3, and whether there is any modification of the receptors in hyper- and hypothyroidism. Lymphocytes were found to possess a high-affinity, limited-capacity binding sites for both T4 and T3. The mean equilibrium affinity constant (Ka) was 2.28 · 1010 ± 0.21 m−1 for T3, and 0.98 · 1010 ± 0.16 m−1 for T4. The mean number of saturable binding sites was 115 for T3, and 102 for T4. The binding capacities and affinities also determined in the lymphocyte nuclei isolated after incubation of the intact cell, were similar to those observed in the intact cells. In competition experiments, labelled T4 was as readily displaced by T3 as by T4 itself, whereas labelled T3 was displaced only by a 40 times higher concentration of T4 than T3. These observations suggest identical receptors for the two hormones and a binding of T4 as such, provided it is not in competition with T3. In lymphocytes from hyperthyroid patients, receptor affinities and numbers remained unchanged. In lymphocytes from hypothyroid patients, the affinity was normal, but the mean number of T3 binding sites was increased to 310 (P < 0.001), to return to normal after a few months of treatment.

Evidence for Presynaptic High Affinity Imidazoline-Specific Binding Sites in the Bovine Brainstem

1999 ◽  
Vol 881 (1 IMIDAZOLINE R) ◽  
pp. 185-188 ◽  
Author(s):  
F. M. J. HEEMSKERK ◽  
M. DONTENWILL ◽  
H. GRENEY ◽  
C. VONTHRON ◽  
P. BOUSQUET
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
High Affinity ◽  
Specific Binding Sites

The High Affinity Calcium Ion Bending Sites of Fibrinogen

1981 ◽  
Author(s):  
Joan Ross ◽  
Graham D Kemp
Keyword(s):  
Calcium Ions ◽  
Binding Sites ◽  
Calcium Ion ◽  
Specific Binding ◽  
Ion Release ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Scatchard Plots ◽  
Systems Studies ◽  
Flow Dialysis

There is considerable evidence that fibrinogen contains a number of strongly bound calcium ions and these appear to have a significant role in the structure and properties of the molecule. Most of the evidence suggests that there are three such strongly bound calcium ions in fibrinogen and each of the two fragments D contains one of these. It has been suggested that the section of the (A) α chain which is the region of the molecule first attacked by plasnin is involved in binding calcium ions. Should this constitute the third site it follows that this calcium ion must link the two (A) α chains and the site may well be destroyed by minimal plasnin attack. The figure of three calcium ions bound, however, must be open to sane doubt due to the difficulty in evaluating data from Scatchard plots prepared from. a system, such as fibrinogen, which contains a number of identical ligands with more than one binding affinity. Accordingly we have developed methods to prepare fibrinogen in as intact a form as possible, and used such fibrinogen in flow dialysis systems. Studies of calcium ion release during proteolytic degradation of fibrinogen lead us to conclude that there are probably only two high affinity, calcium ion specific binding sites in fibrinogen.

Somatostatin binding sites in cytosolic fractions of parietal and non-parietal cells from rabbit fundic mucosa

1985 ◽  
Vol 5 (4) ◽  
pp. 321-328 ◽  
Author(s):  
Eduardo Arilla ◽  
M. Pilar Löpez-Ruiz ◽  
Luis Gonzalez-Guijarro ◽  
Juan C. Prieto
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Parietal Cells ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Fundic Mucosa ◽  
High Affinity ◽  
Specific Binding Sites

Specific binding sites for somatostatin have been found in the cytosolic fractions of both parietal and non-parietal cells from rabbit gastric fundic mucosa. The stoichiometric data suggested the presence of two classes of binding sites in both types of ceils. The number of low-affinity binding sites was significantly higher in parietal cells than in non-parietal cells. The reverse was true for the high-affinity binding sites. However, the affinity of each class of binding sites was similar in the cytosolic fractions of both parietal and non-parietal ceils. It thus appears that low-affinity somatostatin binding sites are mainly located in the parietal ceils whereas the high-affinity sites occur principally in the non-parietal cells.

Binding of [3H]des-Arg9-BK to rabbit anterior mesenteric vein

1982 ◽  
Vol 60 (12) ◽  
pp. 1551-1555 ◽  
Author(s):  
J. Barabé ◽  
C. Babiuk ◽  
D. Regoli
Keyword(s):  
Binding Sites ◽  
De Novo ◽  
Specific Binding ◽  
Kinetic Studies ◽  
Biological Action ◽  
Affinity Constant ◽  
Binding Studies ◽  
Specific Binding Sites ◽  
Pathological Conditions ◽  
Wet Weight

Binding studies of [3H]des-Arg9-BK have been performed on pieces of rabbit anterior mesenteric veins. Kinetic studies have permitted us to evaluate an affinity constant of 1.04 × 10−7 M, which is not so different from the apparent affinity constant determined by bioassay (1.6 × 10−7 M). Furthermore, inhibition of the binding of [3H]des-Arg9-BK with various kinins results in an order of potency of kinins very similar to that observed in the bioassay. Taken together, these results suggest that we are dealing with binding sites which might be the same as those subserving the biological action of des-Arg9-BK (pharmacological receptors). The preincubation of tissues in Krebs' solution brings about an increase of the specific binding from 0.06 pmol/mg of wet weight at time 0 to 0.75 pmol after 24 h; cycloheximide inhibits this increase for at least 6 h. Veins taken from animals treated with LPS, which have shown an increase in sensitivity compared with veins extracted from untreated animals, have a higher number of specific binding sites for [3H]des-Arg9-BK. The results support the hypothesis that the increased response of tissues to des-Arg9-BK is due to the de novo synthesis of receptors for kinins in some experimental and pathological conditions.

scholarly journals Contribution of gangliosides to thyroxin binding with plasma membranes

1995 ◽  
Vol 41 (2) ◽  
pp. 28-30
Author(s):  
T. S. Saatov ◽  
F. Ya. Gulyamova ◽  
G. U. Usmanova
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Specific Binding ◽  
Brain Cell ◽  
Cell Recognition ◽  
High Affinity ◽  
Specific Binding Sites

Besides intracellular receptors of thyroid hormones, specific binding sites for T3 and T4 were detected on plasma membranes (PM) of some cells and a relationship between membrane reception .and lipid composition of membranes shown. The parameters of 125I-T4 binding to highly purified PM of hepatic and cerebral cells of rats were studied. The hepatic and cerebral cellular membranes were found to contain two sites of hormone binding each, one of these sites being characterized by a high affinity and low capacity, and the other by low affinity and a higher binding capacity. The association constant of highly affine site of hepatocyte membranes was found to be higher than that of brain cell membranes. T4 membranous receptors may be significant in the process of cell “recognition" by the hormone. In vivo and in vitro experiments with 125I-T4 and 14C-labeled thyroxin in ganglioside fractions showed appreciable binding of the hormone to Gm3 fraction, this evidently pointing to participation of this, ganglioside in T4 interaction with membrane receptor. It is possible that gangliosides situated on membranous surface are components of or function as receptors.

The Possible Residual Initial Polymerisation Site in Fibrin Plasmic Fragment D-Dimer.

1979 ◽  
Author(s):  
L.R Purves ◽  
G.G. Lindsey
Keyword(s):  
Calcium Ions ◽  
Binding Sites ◽  
Specific Binding ◽  
Molecular Size ◽  
Cross Link ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Partial Dissociation ◽  
Fluorescence Polarisation ◽  
D Dimer

The stable plasmic fragment of fibrin, D-dimer, again becomes susceptible to plasmin digestion if calcium ions are removed. Pragressive cleavage af the C-terminal end of the γ chain occurs releasing a peptide containing the cross link and any biologically attached (F XIII: transglutaminase) dansyl cadaverine. The cleaved peptide binds tightly to the parent malecule but further plasmin digestion can be inhibited by calcium ions. D-dimer has two and D monomer a single high affinity calcium-specific binding sites (Kd 1χ10-5M). The farmation of a noncavalently-linked D-dimer (NCDD) can be shown by the molecular size on malecular sieving and by the retention of molar volume by the dansyl-cadaverine labelled but cleaved D-dimer using fluorescence polarisation. Some partial dissociation occurs at 5°C but reassaciation occurs on return to 35° C. Antibodies to D-dimer have a 5-fold lesser affinity for fibrinogen and D-monomer with our without fragment E present but NCDD gradually loses affinity with extent of cleavage and in time. The interactions of D-dimer with the E domain on fibrinogen and NDSK are dependent on the intact calcium-stabilised γ chain. The inter-D interaction maintaining NCDD could be the residual initial polymerisation site of fibrinogen.

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