scholarly journals Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product.

1995 ◽  
Vol 15 (3) ◽  
pp. 1489-1498 ◽  
Author(s):  
H Nakagama ◽  
G Heinrich ◽  
J Pelletier ◽  
D E Housman
Keyword(s):  
Binding Affinity ◽  
Zinc Finger ◽  
Binding Sites ◽  
Wilms Tumor ◽  
Specific Binding ◽  
Binding Motif ◽  
Urogenital System ◽  
Biochemical Basis ◽  
High Affinity ◽  
Sequence Recognition

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger polypeptide which plays a key role regulating cell growth and differentiation in the urogenital system. Using the whole-genome PCR approach, we searched murine genomic DNA for high-affinity WT1 binding sites and identified a 10-bp motif 5'GCGTGGGAGT3' which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognized by EGR-1 and is also suggested to function as a binding site for WT1, setting up a competitive regulatory loop. To evaluate the underlying biochemical basis for such competition, we compared the binding affinities of WT1 and EGR1 for both sequences. WT1 shows a 20- to 30-fold-higher affinity for the WTE sequence compared with that of the EGR-1 binding motif. Mutational analysis of the WTE motif revealed a significant contribution to binding affinity by the adenine nucleotide at the eighth position (5'GCGTGGGAGT3') as well as by the 3'-most thymine (5'GCGTGGGAGT3'), whereas mutations in either flanking nucleotides or other nucleotides in the core sequence did not significantly affect the specific binding affinity. Mutations within WT1 zinc fingers II to IV abolished the sequence-specific binding of WT1 to WTE, whereas alterations within the first WT1 zinc finger reduced the binding affinity approximately 10-fold but did not abolish sequence recognition. We have thus identified a WT1 target, which, although similar in sequence to the EGR-1 motif, shows a 20- to 30-fold-higher affinity for WT1. These results suggest that physiological action of WT1 is mediated by binding sites of significantly higher affinity than the 9-bp EGR-1 binding motif. The role of the thymine base in contributing to binding affinity is discussed in the context of recent structural analysis.

scholarly journals Adenosine Receptors of Cerebral Microvessels and Choroid Plexus

1986 ◽  
Vol 6 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Rajesh N. Kalaria ◽  
Sami I. Harik
Keyword(s):  
Cerebral Cortex ◽  
Ligand Binding ◽  
Choroid Plexus ◽  
Adenosine Receptors ◽  
Binding Sites ◽  
Specific Binding ◽  
Cerebral Microvessels ◽  
High Affinity ◽  
Receptor Sites ◽  
Ligand Binding Sites

We studied, by ligand binding methods, the two adenosine receptors, A, and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]l-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'- N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity ( Kd ∼ 250 n M) and low-affinity ( Kd ∼ 1–2 μ M) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these microvessels.

scholarly journals Identification of the angiotensin II receptor in rat mesenteric artery

1984 ◽  
Vol 223 (3) ◽  
pp. 659-671 ◽  
Author(s):  
J McQueen ◽  
G D Murray ◽  
P F Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Divalent Cations ◽  
Target Tissue ◽  
Angiotensin Ii Receptor ◽  
High Affinity ◽  
Rat Mesentery ◽  
Rat Mesenteric Artery

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.

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.

Characterization of granulocyte-macrophage colony-stimulating factor receptor on the blast cells of acute myeloblastic leukemia

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 59-66 ◽  
Author(s):  
N Onetto-Pothier ◽  
N Aumont ◽  
A Haman ◽  
C Bigras ◽  
GG Wong ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Receptor Occupancy ◽  
Gm Csf ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Macrophage Colony Stimulating Factor ◽  
Affinity Receptor ◽  
Acid Wash ◽  
Macrophage Colony

Abstract Iodinated granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to document the specific binding of GM-CSF to all acute myeloblastic leukemia (AML) samples examined in the present study. There was some heterogeneity in the number of GM-CSF binding sites per cell. To determine whether the low level of binding to some patient samples may be attributed to receptor occupancy by an endogenous source of GM-CSF, we devised an acid wash procedure that could remove surface- bound GM-CSF without affecting receptor properties. We thus document that GM-CSF specific binding to AML blasts before or after acid wash was the same, indicating that the observed heterogeneity in binding is not the result of receptor occupancy by an endogeneous source of GM- CSF. Saturation analyses are in favor of the presence of two classes of binding sites on AML blasts: a high-affinity receptor that binds GM-CSF with a dissociation constant (kd) of 3 to 73 pmol/L and a second class of low-affinity receptor that binds GM-CSF with a kd of 1 to 10 nmol/L. Binding studies with two established cell lines KG-1, and IRCM-8 also showed the presence of two classes of binding sites with high and low affinities. Analysis of GM-CSF titration curves in culture indicate that the median effective concentration required for stimulation of blast colony formation (EC50 = 5–36 pmol/L) were in the range of the kd of the high-affinity binding site, suggesting that this high-affinity binding site mediates the proliferative response.

Prostaglandin E2 binding sites in porcine oxyntic mucosa: effects of salicylates

1986 ◽  
Vol 64 (5) ◽  
pp. 515-520 ◽  
Author(s):  
B. L. Tepperman ◽  
B. D. Soper
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Prostaglandin E ◽  
Affinity Binding ◽  
Variable Degree ◽  
Single Class ◽  
Oxyntic Mucosa ◽  
High Affinity Binding ◽  
Mucosal Ulceration ◽  
High Affinity

These studies were designed to examine the changes in the characteristics of prostaglandin E2 (PGE2) binding to porcine oxyntic mucosa in the response to oral ingestion of salicylates. Either acetylsalicylic acid (ASA) or salicylic acid (SA) was administered to conscious pigs (100 mg/kg in 30 mL of an equimolar concentration of NaHCO3) once a day for 1, 3, 10, or 20 days. In control experiments a similar volume of 0.3 M NaHCO3 was administered for similar durations. Mucosal ulceration and the characteristics of the binding of [3H]PGE2 to a 30 000 × g membrane preparation of oxyntic mucosa were examined. Generation of mucosal PGE2 was measured by radioimmunoassay. ASA treatment resulted in an increase in the number and severity of mucosal ulcers and a decrease in PGE2 levels within the first treatment day. By day 20 the degree of ulceration had decreased in spite of a persistent reduction of mucosal PGE2 generation. A variable degree of ulceration was observed in SA-treated animals. In control animals only a single class of binding sites for [3H]PGE2 was evident. After 3 days of ASA treatment a second class of binding sites with a high affinity dissociation constant appeared. There was a decrease in the high affinity binding of [3H]PGE2 after 20 days of ASA ingestion. Low affinity binding was not altered. ASA treatment resulted in a significant increase in specific binding capacities for both families of binding sites. SA treatment did not consistently alter PGE2 binding characteristics from control at any time period studied. These data suggest that SA treatment results in a small degree of mucosal damage in the absence of a significant reduction in tissue generation of PGE2 or changes in PGE2 binding. Damage in response to ASA ingestion was associated with a reduction in both endogenous synthesis of PGE2 and an increase in the concentration of both low and high affinity binding sites for PGE2. The reduction in mucosal ulceration on day 20 in spite of depressed endogenous PGE2 coincides with an increase in PGE2 binding.

scholarly journals The accumulation and compartmentalization of isometamidium chloride in Trypanosoma congolense, monitored by its intrinsic fluorescence

1995 ◽  
Vol 312 (1) ◽  
pp. 319-327 ◽  
Author(s):  
J M Wilkes ◽  
A S Peregrine ◽  
D Zilberstein
Keyword(s):  
Binding Sites ◽  
Thermal Denaturation ◽  
Specific Binding ◽  
Trypanosoma Congolense ◽  
Emission Peak ◽  
Intrinsic Fluorescence ◽  
Drug Uptake ◽  
Membrane Transporter ◽  
High Affinity ◽  
Dnase 1

Interaction of the trypanocide isometamidium chloride with components of Trypanosoma congolense results in characteristic shifts in the intrinsic fluorescence of the drug. The specificity of this interaction was investigated by analysing the effects of various physicochemical manipulations on its fluorescence properties. The characteristic shifts involved a preferential increase in the intensity of one emission peak over the other, resulting in a systematic increase in the ratio of fluorescence intensities. These effects were apparently due to constraints on fluorophore free rotation in the solution (that is, viscosity). Purified DNA produced similar effects in a saturable manner displaying high affinity for the drug, indicating that the constraint involves binding of the drug to high-affinity binding sites within the DNA. Such binding sites were demonstrated in lysates derived from trypanosomal cells. The binding sites were associated with macromolecular species (M(r) > 12000), and were partly disrupted by thermal denaturation and proteolysis. Treatment with DNase 1 produced high levels of disruption of the binding sites (> 85%), indicating an involvement of DNA in the binding. BSA demonstrated weak non-specific binding of the drug. Entry of drug into live trypanosomal cells (monitored by 14C-labelled drug uptake) was paralleled by fluorescence shifts observed under comparable conditions of drug concentration and buffer conditions. Both systems (fluorescence shifts and accumulation of labelled drug) indicated the presence of a saturable membrane transporter with high affinity for the drug. We conclude that monitoring the fluorescence shifts of isometamidium constitutes a sensitive and highly specific probe for entry of the drug into trypanosomal cells, thereby enabling resolution of the transport events involved.

scholarly journals Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger.

1991 ◽  
Vol 11 (4) ◽  
pp. 1777-1784 ◽  
Author(s):  
Y D Halvorsen ◽  
K Nandabalan ◽  
R C Dickson
Keyword(s):  
Base Pair ◽  
Specific Binding ◽  
Kluyveromyces Lactis ◽  
Affinity Binding ◽  
Transcription Activator ◽  
High Affinity Binding ◽  
High Affinity ◽  
Sequence Recognition ◽  
Broad Region ◽  
Dna Helix

The LAC9 protein of Kluyveromyces lactis is a transcriptional regulator of genes in the lactose-galactose regulon. To regulate transcription, LAC9 must bind to 17-bp upstream activator sequences (UASs) located in front of each target gene. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae, and the two proteins must bind DNA in a very similar manner. In this paper we show that high-affinity, sequence-specific binding by LAC9 dimers is mediated primarily by 3 bp at each end of the UAS: [Formula: see text]. In addition, at least one half of the UAS must have a GC or CG base pair at position 1 for high-affinity binding; LAC9 binds preferentially to the half containing the GC base pair. Bases at positions 2, 3, and 4 in each half of the UAS make little if any contribution to binding. The center base pair is not essential for high-affinity LAC9 binding when DNA-binding activity measured in vitro. However, the center base pair must play an essential role in vivo, since all natural UASs have 17, not 16, bp. Hydroxyl radical footprinting shows that a LAC9 dimer binds an unusually broad region on one face of the DNA helix. Because of the data, we suggest that LAC9 contacts positions 6, 7, and 8, both plus and minus, of the UAS, which are separated by more than one turn of the DNA helix, and twists part way around the DNA, thus protecting the broad region of the minor groove between the major-groove contacts.

Characterization and localization of oxytocin receptors in the uterus and oviduct of the non-pregnant ewe using an iodinated receptor antagonist

1991 ◽  
Vol 128 (2) ◽  
pp. 187-NP ◽  
Author(s):  
V. J. Ayad ◽  
S. E. F. Guldenaar ◽  
D. C. Wathes
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Oxytocin Receptor ◽  
Binding Characteristics ◽  
High Affinity ◽  
Oxytocin Receptors ◽  
Secretory Glands ◽  
Pregnant Ewe ◽  
Autoradiographic Technique ◽  
Specific Labelling

ABSTRACT Some of the binding characteristics of a novel oxytocin receptor ligand 125I-labelled [1-(β-mercapto-β, β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2,Thr4,Orn8,Tyr9-NH2]-vasotocin ([125I]OTA) have been determined in the sheep uterus. The compound was subsequently used for the autoradiographic localization of oxytocin receptors in the uterus and oviduct of the ewe. Specific binding of [125I]OTA to crude membrane fractions of ovine endometrium was time-dependent and was unaffected by the addition of cations to incubation media. Endometrial membranes contained a single population of saturable, high-affinity binding sites for the iodinated ligand (dissociation constant (Kd) 0·23±0·08 nmol/l) and unlabelled oxytocin competed with [125I]OTA for binding sites with high affinity (Kd 1·29±0·4 nmol/l) in the presence of Mg2+ In contrast, unlabelled OTA was able to compete with high affinity (Kd 1·13±0·16 nmol/l) in the absence of cation. Competition studies with a number of oxytocin analogues and related peptides and the tissue distribution of [125I]OTA binding sites also indicated that [125I]OTA bound to the ovine oxytocin receptor. This was further validated by autoradiographic studies which showed specific labelling with [125I]OTA to be greater to uterus and oviduct obtained from ewes which had been killed within 2 days of oestrus than to similar tissue from ewes killed during the luteal phase. In both the ampullary and isthmic regions of the oviduct and the myometrium, [125I]OTA binding sites were confined to smooth muscle. Endometrial binding sites for [125I]OTA were consistently located on the luminal epithelium and epithelial cells lining secretory glands. In addition, in one ewe which had been killed 2 days after cloprostenol treatment, stromal cells were labelled in a caruncular region of the endometrium. The consistency of this observation between similar animals remains to be determined. The autoradiographic technique demonstrated appears sufficiently sensitive to allow further studies into the distribution of the endometrial oxytocin receptor throughout the oestrous cycle, and into its regulation at luteolysis and during the establishment of pregnancy. Journal of Endocrinology (1991) 128, 187–195

Abstract P622: Characterization of 125-I-Angiotensin (1-7) Binding to Rat Kidney

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Filipe F Conti ◽  
Andrea Linares ◽  
Leena E Couling ◽  
Mariana Morris ◽  
Katia De Angelis ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Affinity ◽  
Binding Sites ◽  
Specific Activity ◽  
Specific Binding ◽  
Rat Kidney ◽  
Albino Rats ◽  
Angiotensin Peptides ◽  
Putative Receptor ◽  
Wet Weight

Despite the plethora of data indicating beneficial effects of angiotensin (1-7) (Ang 1-7) on the cardiovascular system, its putative receptor, Mas, has not been characterized in tissue membrane preparations other than single concentration demonstrations of the localization of 125 I-Ang 1-7 binding sites in rat kidney. This does not indicate the specificity of 125 I-Ang 1-7 binding nor does it indicate the actual densities of the binding sites, i.e., B max (fmoles/mg tissue), or dissociation constant (K D ) to indicate binding affinity of 125 I-Ang 1-7 for its putative receptor. To characterize 125 I-Ang 1-7 binding in the kidney we prepared a low specific activity, monoradioiodinated Ang 1-7 using a 1:19 mix of 125 iodine : 127 iodine which allows for assessment of the B max and K D with concentrations of radioligand up to 100 nM. Frozen kidneys from adult male albino rats were dissected and homogenized in water and the membranes were precipitated by centrifugation at 48 kxG. Membranes were resuspended in Tris:MgCl 2 (50:1) pH 7.2 and incubated with 12 concentrations of 125/127 I-Ang 1-7 ranging from ~3-100 nM for 30 min at 22 C, after which bound 125/127 I-Ang 1-7 was resolved from unbound 125/127 I-Ang 1-7 by filtration and measured with a gamma counter. Specific binding (defined as 100 μM Ang 1-7 displaceable binding) of 125/127 I-Ang 1-7 showed a moderate binding affinity (K D = 14.7 ± 1.8 nM) and binding site density (B max = 24.5 ± 9.9 fmoles/mg initial wet weight). The B max value tended to be lower than that in the liver (B max = 62.3 ± 20.1 fmoles/mg initial wet weight) and the K D value was significantly greater (lower affinity) than that in the liver ( K D = 5.7 ± 0.6 nM, p = 0.0085). Of note, competition for 125/127 I-Ang 1-7 binding Ang 1-7 indicated that the IC 50 for Ang 1-7 competition for 125/127 I-Ang 1-7 binding was 42.5 μM. Moreover, the ability of a variety of angiotensin peptides to inhibit 125/127 I-Ang 1-7 binding at 100 μM, Ang 1-7 was less potent that the other angiotensin peptides: Ang III > Ang II > Ang I ~ Ang IV > Ang 2-7 > Ang 1-7 ~ Ang 3-7. These studies suggest that the binding site for 125/127 I-Ang 1-7 is not specific for the putative Ang 1-7 receptor mas, and may represent a low affinity binding to the AT 1 or AT 2 receptor

Sign in / Sign up

Export Citation Format

Share Document