scholarly journals Using electrophoretic mobility shift assays to measure equilibrium dissociation constants: GAL4-p53 binding DNA as a model system

2012 ◽  
Vol 40 (6) ◽  
pp. 383-387 ◽  
Author(s):  
Michael A. Heffler ◽  
Ryan D. Walters ◽  
Jennifer F. Kugel‡
Keyword(s):  
Electrophoretic Mobility ◽  
Dissociation Constants ◽  
Model System ◽  
Mobility Shift ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation ◽  
Electrophoretic Mobility Shift Assays

scholarly journals Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity

2004 ◽  
Vol 186 (18) ◽  
pp. 6254-6264 ◽  
Author(s):  
Peggy Freede ◽  
Sabine Brantl
Keyword(s):  
Binding Affinity ◽  
Copy Number ◽  
Dissociation Constants ◽  
Mobility Shift ◽  
Copy Numbers ◽  
Maximal Binding ◽  
Equilibrium Dissociation Constants ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of the repR promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5′-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 μM, respectively. In this work, we used a SELEX procedure to study copR operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated copR operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3′ positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated.

Altered plasma catecholamines and numbers of α- and β-adrenergic receptors in platelets and leucocytes in hyperthyroid patients normalized under antithyroid treatment

1985 ◽  
Vol 110 (1) ◽  
pp. 75-82 ◽  
Author(s):  
Dieter Ratge ◽  
Sabine Hansel-Bessey ◽  
Hermann Wisser
Keyword(s):  
Dissociation Constants ◽  
Plasma Catecholamines ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Plasma Norepinephrine ◽  
Thyroid State ◽  
Antithyroid Treatment ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation ◽  
Hyperthyroid Patients

Abstract. We measured plasma catecholamines, α- and β-adrenoreceptor numbers and the accumulation of cyclic adenosine monophosphate (cAMP) in the unstimulated state and in response to 10 μmol/l (-) isoproterenol in blood cells from 29 euthyroid controls and from 18 patients with spontaneous hyperthyroidism. In the thyrotoxic patients plasma norepinephrine (1.14 ± 0.5 nmol/l) and epinephrine (0.3 ±0.14 nmol/l) were significantly decreased compared with plasma norepinephrine (1.87 ± 0.7 nmol) and epinephrine (0.41 ± 0.19 nmol/l) in the controls (P < 0.01 and P < 0.05, respectively) and the values obtained in subjects rendered euthyroid by antithyroid treatment (P < 0.001, respectively). α-adrenoceptor density in platelet membranes obtained from patients in the hyperthyroid state (114 ± 38 sites per cell) was significantly decreased when compared with controls (159 ± 48 sites per cell, P < 0.01) and the values from patients under effective antithyroid treatment (136 ± 35 sites per cell, P < 0.01). On the contrary, a significant increase in β-adrenoceptor density in mononuclear leucocyte (MNL) membranes was found in hyperthyroid patients (1751 ± 237 sites/cell) when compared with controls (1510 ± 351 sites/cell, P < 0.05) and the same patients following antithyroid treatment (1455 ± 260 sites/cell, P < 0.001). The equilibrium dissociation constants (KD) did not change in hyperthyroidism. Basal cAMP concentrations in MNL were higher in untreated thyrotoxicosis (45 ± 18 pmol/106 cells/10 min) than in patients in the euthyroid state (35 ± 9 pmol/106 cells/10 min, P < 0.05). Our data support the hypothesis that the balance of α- and β-adrenoceptors depends on the thyroid state. However, before the reputed catecholamine supersensitivity in hyperthyroid man can be accepted, the relationship between alterations in adrenoceptors and the biological responsiveness to catecholamines has to be demonstrated in different human tissues.

scholarly journals Circular dichroism determination of class I MHC-peptide equilibrium dissociation constants

1997 ◽  
Vol 6 (8) ◽  
pp. 1771-1773 ◽  
Author(s):  
Chantal S. Morgan ◽  
James M. Holton ◽  
Barry D. Olafson ◽  
Pamela J. Bjorkman ◽  
Stephen L. Mayo
Keyword(s):  
Circular Dichroism ◽  
Dissociation Constants ◽  
Class I ◽  
Class I Mhc ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation ◽  
Circular Dichroïsm

Preparation and Characterization of Paclitaxel Imprinted Silica Nanoparticles

2011 ◽  
Vol 399-401 ◽  
pp. 1894-1897
Author(s):  
Jian Hua Li ◽  
Zong Jian Zheng ◽  
Shao Ping Fu ◽  
Jing Bo Zhu
Keyword(s):  
Silica Nanoparticles ◽  
Dissociation Constants ◽  
Sol Gel ◽  
Scatchard Analysis ◽  
Binding Properties ◽  
Imprinted Polymers ◽  
Sol Gel Process ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation

Highly selective molecularly imprinted layer-coated silica nanoparticles for paclitaxel were synthesized by molecular imprinting technique with a sol–gel process on the supporter of silica nanoparticles. The morphology of the obtained polymers was characterized by scanning electron microscopy (SEM). The binding properties of the imprinted polymers were evaluated through the equilibrium rebinding experiments. Scatchard analysis revealed that two classes of binding sites were formed in the imprinted polymers with equilibrium dissociation constants of 0.0509 g•L-1and 0.0094 g•L-1, respectively. Paclitaxel and its analogue were employed for selectivity tests. The results indicated that the imprinted polymers exhibited good selectivity and specificity toward paclitaxel.

Adrenal medullary regulation of rat renal cortical adrenergic receptors

1987 ◽  
Vol 253 (5) ◽  
pp. F1063-F1067
Author(s):  
P. R. Sundaresan ◽  
M. M. Guarnaccia ◽  
J. L. Izzo
Keyword(s):  
Adrenal Medulla ◽  
Adrenergic Receptors ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Physiological State ◽  
Plasma Corticosterone ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation

The role of the adrenal medulla in the regulation of renal cortical adrenergic receptors was investigated in renal cortical particulate fractions from control rats and rats 6 wk after adrenal demedullation. The specific binding of [3H]prazosin, [3H]rauwolscine, and [125I]iodocyanopindolol were used to quantitate alpha 1-, alpha 2-, and beta-adrenergic receptors, respectively. Adrenal demedullation increased the concentration of all three groups of renal adrenergic receptors; maximal number of binding sites (Bmax, per milligram membrane protein) for alpha 1-, and alpha 2-, and beta-adrenergic receptors were increased by 22, 18.5, and 25%, respectively (P less than 0.05 for each). No differences were found in the equilibrium dissociation constants (KD) for any of the radioligands. Plasma corticosterone and plasma and renal norepinephrine levels were unchanged, whereas plasma epinephrine was decreased 72% by adrenal demedullation (P less than 0.01); renal cortical epinephrine was not detectable in control or demedullated animals. Our results suggest that, in the physiological state, the adrenal medulla modulates the number of renal cortical adrenergic receptors, presumably through the actions of a circulating factor such as epinephrine.

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