Effect of receptor occupancy on [3H]dexamethasone binding to circulating leukocytes

1980 ◽  
Vol 94 (3) ◽  
pp. 376-380 ◽  
Author(s):  
Tohru Murakami ◽  
David D. Brandon ◽  
D. Lynn Loriaux ◽  
Mortimer B. Lipsett
Keyword(s):  
Glucocorticoid Receptor ◽  
Binding Capacity ◽  
Receptor Occupancy ◽  
Binding Study ◽  
Scatchard Plot ◽  
Total Binding ◽  
Plot Analysis ◽  
Scatchard Plot Analysis ◽  
Receptor Concentration

Abstract. To determine the effect of steroid occupancy of the glucocorticoid receptor on the measurement of total receptor concentration using Scatchard plot of [3H]dexamethasone (Dex) binding to circulating leukocytes, leukocytes were incubated with 10−7m cortisol or 10−9m Dex prior to [3H]Dex binding study. Total binding capacity calculated from Scatchard plot analysis of [3H]Dex binding was not significantly reduced after pre-incubation with cortisol. However, total binding capacity was significantly lower after pre-incubation with Dex. These data suggest that total glucocorticoid receptor concentration can be measured from a 3 h incubation of leukocytes obtained from untreated patients.

Scatchard Plot Analysis of Ligand-Erythrocyte Interactions

1978 ◽  
Vol 67 (9) ◽  
pp. 1344-1345 ◽  
Author(s):  
D.L. Parsons ◽  
J.J. Vallner
Keyword(s):  
Scatchard Plot ◽  
Plot Analysis ◽  
Scatchard Plot Analysis

Evidence for p55–p75 heterodimers in the absence of IL-2 from Scatchard plot analysis

1992 ◽  
Vol 4 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Byron Goldstein ◽  
David Jones ◽  
Ioannis G. Kevrekidis ◽  
Alan S. Perelson
Keyword(s):  
Scatchard Plot ◽  
Plot Analysis ◽  
Scatchard Plot Analysis

Binding of mitochondrial malate dehydrogenase to mitoplasts

1979 ◽  
Vol 57 (6) ◽  
pp. 662-665 ◽  
Author(s):  
Paula M. Strasberg ◽  
Keith A. Webster ◽  
Hasmukh V. Patel ◽  
Karl B. Freeman
Keyword(s):  
Malate Dehydrogenase ◽  
Rat Liver ◽  
Specific Binding ◽  
Liver Mitochondria ◽  
Scatchard Plot ◽  
Rat Liver Mitochondria ◽  
Isolated Mitochondria ◽  
Plot Analysis ◽  
Scatchard Plot Analysis ◽  
Mitochondrial Malate Dehydrogenase

The binding of 14C-labelled bovine and porcine malate dehydrogenase (EC 1.1.1.37) to rat liver mitochondria and mitoplasts was examined. The bovine enzyme was found to associate nonspecifically with isolated mitochondria and sonicated mitoplasts. Scatchard plot analysis suggested a specific binding to mitoplasts of the order of 5 pmol malate dehydrogenase per milligram of mitoplast protein. Porcine malate dehydrogenase dimer but not monomer exhibited a similar binding. The results are discussed in relation to the mechanism of uptake of the enzyme by mitochondria after synthesis on cytosolic ribosomes.

Oestrogen receptors in endometrial cytosol of gilts on days 10-13 of oestrous cycle and pregnancy

1994 ◽  
Vol 42 (3) ◽  
pp. 217-223
Author(s):  
J. Van Der Meulen ◽  
F.A. Helmond ◽  
C.P.J. Oudenaarden
Keyword(s):  
Oestrous Cycle ◽  
Endometrial Tissue ◽  
Scatchard Plot ◽  
Oestrogen Receptors ◽  
Reproductive State ◽  
Dextran Coated Charcoal ◽  
Plot Analysis ◽  
Scatchard Plot Analysis

Cytosolic oestrogen receptors (ER(c)) were determined with a Dextran-coated charcoal assay and Scatchard plot analysis in endometrial tissue of non-pregnant and pregnant gilts on Days 10-13 after standing oestrus. The Kd of the ER(c) was 0.40 +/- 0.04 nM (mean +/- SEM) and was not affected by day or reproductive state. The endometrial ER(c) concentration was affected by day and reproductive state (P

scholarly journals Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

2008 ◽  
Vol 22 (8) ◽  
pp. 1754-1766 ◽  
Author(s):  
Weiwei Chen ◽  
Thoa Dang ◽  
Raymond D. Blind ◽  
Zhen Wang ◽  
Claudio N. Cavasotto ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Divalent Cations ◽  
Phosphorylation Site ◽  
Transcriptional Response ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Interacting Protein

Abstract The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

Temperature-dependent binding of cyclosporine to an erythrocyte protein.

1987 ◽  
Vol 33 (4) ◽  
pp. 481-485 ◽  
Author(s):  
R P Agarwal ◽  
G A Threatte ◽  
R A McPherson
Keyword(s):  
Binding Constant ◽  
Binding Capacity ◽  
Equilibrium Dialysis ◽  
Temperature Dependent ◽  
Fixed Amount ◽  
Competitive Binding Assay ◽  
Plot Analysis ◽  
Binding Data ◽  
Saturation Capacity ◽  
Increasing Temperature

Abstract In this competitive binding assay to measure endogenous binding capacity for cyclosporine (CsA) in erythrocyte lysates, a fixed amount of [3H]CsA plus various concentrations of unlabeled CsA is incubated with aliquots of a test hemolysate. Free CsA is then adsorbed onto charcoal and removed by centrifugation; CsA complexed with a cyclosporine-binding protein (CsBP) remains in the supernate. We confirmed the validity of this charcoal-separation mode of binding analysis by comparison with equilibrium dialysis. Scatchard plot analysis of the results at 4 degrees C yielded a straight line with slope corresponding to a binding constant of 1.9 X 10(7) L/mol and a saturation capacity of approximately 4 mumol per liter of packed erythrocytes. Similar analysis of binding data at 24 degrees C and 37 degrees C showed that the binding constant decreased with increasing temperature, but the saturation capacity did not change. CsBP was not membrane bound but appeared to be freely distributed within erythrocytes. 125I-labeled CsA did not complex with the erythrocyte CsBP. Several antibiotics and other drugs did not inhibit binding between CsA and CsBP. These findings may explain the temperature-dependent uptake of CsA by erythrocytes in whole blood and suggest that measurement of CsBP in erythrocytes or lymphocytes may help predict therapeutic response or toxicity after administration of CsA.

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