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.

Regulation of angiotensin II binding sites in neuronal cultures by catecholamines

1989 ◽  
Vol 257 (4) ◽  
pp. C706-C713 ◽  
Author(s):  
L. M. Myers ◽  
C. Sumners
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Phorbol Esters ◽  
De Novo ◽  
Specific Binding ◽  
Physiological Mechanism ◽  
Neuronal Cultures ◽  
Ang Ii ◽  
Specific Binding Sites ◽  
Stimulation Of

Previous studies determined that direct activation of protein kinase C (PKC) with phorbol esters increases the number of angiotensin II (ANG II)-specific binding sites in neuronal cultures prepared from the hypothalamus and brain stem of 1-day-old rats. In the physiological situation, PKC is activated by diacylglycerol, which can be produced by multiple pathways, such as stimulation of inositol phospholipid (IP) hydrolysis, phosphatidylcholine hydrolysis, or by de novo synthesis. In the present study we have examined whether stimulation of IP hydrolysis, and presumably activation of PKC, can mimic the actions of phorbol esters on ANG II-specific binding. We have incubated neuronal cultures with agents that increase IP hydrolysis and have determined the effects on ANG II-specific binding. Incubation of neuronal cultures with norepinephrine (NE) at concentrations (greater than 5 microM) and for times (15-60 min) that cause large increases in IP hydrolysis caused increases in the number of ANG II-specific binding sites, mimicking the actions of phorbol esters. The return of IP hydrolysis to control values was associated with a return of ANG II-specific binding to control levels. The upregulatory action of NE was abolished by prazosin, demonstrating the involvement of alpha 1-adrenergic receptors. In addition, this effect was blunted by the PKC antagonist H 7, suggesting PKC involvement in the response. Thus we have determined a potential physiological mechanism by which stimulation of IP hydrolysis by NE, and possible subsequent activation of PKC, leads to upregulation of ANG II-specific binding sites in neuronal cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic detection of kinin receptors in the human medulla of control, hypertensive, and diabetic donors

2002 ◽  
Vol 80 (4) ◽  
pp. 249-257 ◽  
Author(s):  
Hudson de Sousa Buck ◽  
Brice Ongali ◽  
Gaétan Thibault ◽  
Charles J Lindsey ◽  
Réjean Couture
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
Inferior Olivary Nucleus ◽  
Binding Studies ◽  
Specific Binding Sites ◽  
Kinin Receptors ◽  
Medullary Nuclei ◽  
Inferior Olivary

Kinins have been elected to the status of central neuromediators. Their effects are mediated through the activation of two G-protein-coupled receptors, denoted B1 and B2. Functional and binding studies suggested that B1 and B2 receptors are upregulated in the medulla and spinal cord of hypertensive and diabetic rats. The aim of this study was to localize and quantify kinin receptors in post-mortem human medulla obtained from normotensive, hypertensive, and diabetic subjects, using in vitro receptor autoradiography with the radioligands [125I]HPP-HOE140 (B2 receptor) and [125I]HPP[des-Arg10]-HOE140 (B1 receptor). Data showed specific binding sites for B2 receptor (0.4–1.5 fmol/mg tissue) in 11 medullary nuclei from 4 control specimens (paratrigeminal > ambiguus > cuneate, gelatinous layer of the caudal spinal trigeminal nucleus > caudal and interpolar spinal trigeminal, external cuneate, solitary tract > hypoglossal > gracile > inferior olivary nuclei). Increased density of B2 receptor binding sites was observed in seven medullary nuclei of four hypertensive specimens (paratrigeminal > external cuneate > interpolar and caudal spinal trigeminal, gracile, inferior olivary > hypoglossal nuclei). B2 receptor binding sites were seemingly increased in the same medullary nuclei of two diabetic specimens. Specific binding sites for B1 receptor (1.05 and 1.36 fmol/mg tissue) were seen only in the inferior olivary nucleus in two out of the ten studied specimens. The present results support a putative role for kinins in the regulation of autonomic, nociceptive, and motor functions at the level of the human medulla. Evidence is also provided that B2 receptors are upregulated in medullary cardiovascular centers of subjects afflicted of cardiovascular diseases.Key words: bradykinin, hypertension, diabetes, human brain.

The binding of [3H]mepyramine to histamine H1 receptors in monkey brain

1985 ◽  
Vol 63 (6) ◽  
pp. 756-759 ◽  
Author(s):  
B. Bielkiewicz ◽  
D. A. Cook
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Histamine Receptor ◽  
Detailed Examination ◽  
Mammalian Brain ◽  
Binding Studies ◽  
Peripheral Tissues ◽  
Monkey Brain ◽  
Specific Binding Sites ◽  
Histamine H1 Receptors

Several laboratories have reported ligand binding studies using radioactive histamine H1 antagonists to label the H1 receptors in mammalian brain. We have extended these studies to a detailed examination of the binding of [3H]mepyramine to monkey brain and have shown that the distribution is similar to that in man, with specific binding sites being concentrated in the frontal cortex with relatively low binding to the pons and basal ganglia. The binding shows a single saturable component with a KD of about 1 nM and a Hill plot slope close to unity. These observations are the same for all structures tested. Comparison with data from other laboratories suggests that in this species, the histamine receptor is the same as that in peripheral tissues. From Ki values for various ligands and comparison of KD estimates in other species, the receptor seems to be essentially identical to the H1 receptor in central and peripheral tissues of the guinea pig and also to that in human brain. The rat and possibly the dog have minor differences from the monkey in terms of KD values for [3H]mepyramine binding.

scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2020 ◽  
Vol 48 (16) ◽  
pp. 8914-8926
Author(s):  
Erin E Cutts ◽  
J Barry Egan ◽  
Ian B Dodd ◽  
Keith E Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

Abstract The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

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 A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2019 ◽  
Author(s):  
Erin Cutts ◽  
J. Barry Egan ◽  
Ian Dodd ◽  
Keith Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

AbstractThe Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA, and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl’s repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

scholarly journals The inhibition of mitochondrial calcium transport by lanthanides and Ruthenium Red

1974 ◽  
Vol 140 (2) ◽  
pp. 143-155 ◽  
Author(s):  
Ken C. Reed ◽  
Fyfe L. Bygrave
Keyword(s):  
Binding Sites ◽  
Calcium Transport ◽  
Specific Binding ◽  
Protein A ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Calcium ◽  
Binding Studies ◽  
Specific Binding Sites

An EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-quench technique was developed for measuring initial rates of 45Ca2+ transport by rat liver mitochondria. This method was used in conjunction with studies of Ca2+-stimulated respiration to examine the mechanisms of inhibition of Ca2+ transport by the lanthanides and Ruthenium Red. Ruthenium Red inhibits Ca2+ transport non-competitively with Ki 3×10-8m; there are 0.08nmol of carrier-specific binding sites/mg of protein. The inhibition by La3+ is competitive (Ki=2×10-8m); the concentration of lanthanide-sensitive sites is less than 0.001nmol/mg of protein. A further difference between their modes of action is that lanthanide inhibition diminishes with time whereas that by Ruthenium Red does not. Binding studies showed that both classes of inhibitor bind to a relatively large number of external sites (probably identical with the ‘low-affinity’ Ca2+-binding sites). La3+ competes with Ruthenium Red for most of these sites, but a small fraction of the bound Ruthenium Red (less than 2nmol/mg of protein) is not displaced by La3+. The results are discussed briefly in relation to possible models for a Ca2+ carrier.

Sign in / Sign up

Export Citation Format

Share Document