hCG-Leydig cell functional binding kinetics: threshold and nonlinearity in response

1980 ◽  
Vol 238 (3) ◽  
pp. E293-E302
Author(s):  
W. R. Moyle ◽  
M. Netburn ◽  
A. E. Cosgrove ◽  
J. Krieger ◽  
O. P. Bahl
Keyword(s):  
Activation Energy ◽  
Cyclic Amp ◽  
Leydig Cell ◽  
Binding Sites ◽  
Receptor Occupancy ◽  
Binding Kinetics ◽  
Receptor Complex ◽  
Cyclic Amp Accumulation ◽  
Kinetics Of ◽  
Testosterone Synthesis

Functional kinetic methods, developed to measure the interaction of human chorionic gonadotropin (hCG) with rat Leydig-cell receptors, appear to be useful tools for correlating response with receptor occupancy. In the functional procedures, hCG was allowed to bind to the cells (period I), the free hormone was removed by washing and/or antiserum treatment (period II), and the response of the cells was measured at 37 degrees C (period III). Once initiated, the response to hCG was stable throughout period III. Assuming a one-to-one relationship between occupancy and response during period III, we estimated the rate of association to be 10(8) M-1/min at 37 degrees C with an activation energy of 14-17 kcal/mol. Removal of sialic acid from hCG increased this rate; removal of other carbohydrate residues decreased it. Similar values for the kinetics of binding were observed when either steroidogenesis or cyclic AMP accumulation was measured, suggesting that the same receptor may mediate both processes. Use of either functional or direct (i.e., 125I-labeled hCG) methods to estimate response as a function of occupancy gave equal results, suggesting that most binding sites were coupled to a response. Response was nonlinearly coupled to occupany. Threshold amounts of hormone-receptor complex (0.1% total receptors testosterone synthesis; 2.7% total receptors cyclic AMP accumulation) were required to induce any response. Increased stimulation required progressively larger increments of receptor occupancy. The threshold was inversely proportional to the efficacy of the hCG derivative used and was reduced by the presence of isobutylmethylxanthine.

Coexistence of novel amylin-binding sites with calcitonin receptors in human breast carcinoma MCF-7 cells

1997 ◽  
Vol 155 (3) ◽  
pp. 423-431 ◽  
Author(s):  
U Zimmermann ◽  
B Fluehmann ◽  
W Born ◽  
JA Fischer ◽  
R Muff
Keyword(s):  
Breast Carcinoma ◽  
Cyclic Amp ◽  
Binding Sites ◽  
Human Breast Carcinoma ◽  
Human Breast ◽  
T47d Cells ◽  
Amino Terminal ◽  
Cyclic Amp Accumulation ◽  
Human Amylin ◽  
Mcf 7

Amylin, calcitonin (CT) and calcitonin gene-related peptide (CGRP) share limited structural homology including amino-terminal ring structures linked by a disulfide bridge and amidated carboxy-termini. Here, we have compared [125I]Bolton-Hunter-[Lys1] rat amylin ([125I]amylin) binding and the stimulation of cyclic AMP accumulation by human (h) amylin, hCT and hCGRP-I in the human breast carcinoma cell lines MCF-7 and T47D, which predominantly express hCT1a and hCT1b receptor isoforms (hCTR1a, hCTR1b) at a similar total number of hCT-binding sites. In MCF-7 cells, half-maximal inhibition (IC50) of [125I]amylin binding by human amylin was observed at 3.6 +/- 0.8 nM (n = 6). hCT and hCGRP-I displaced [125I]amylin binding with 22 and 66 times higher IC50. [125I]hCT binding was inhibited by hCT with an IC50 of 8.1 +/- 1.9 nM (n = 5), and human amylin and hCGRP-I were over 100 times less potent. In T47D cells, on the other hand, specific binding of [125I]amylin was not observed, but hCT inhibited [125I]hCT binding with an IC50 of 3.2 +/- 0.4 nM (n = 3), and human amylin and hCGRP-I had over 200 times higher IC50. In MCF-7 cells, half-maximal stimulation (EC50) of cyclic AMP accumulation by human amylin, hCT and hCGRP-I occurred at 1.4 +/- 0.2, 1.7 +/- 0.4 and 6.3 +/- 1.3 nM respectively. In T47D cells, the EC50 of hCT was 0.32 +/- 0.02 nM (n = 3), and 30- and 1900-fold higher with human amylin and hCGRP-I. In conclusion, the expression of hCTR1a and hCTR1b and [125I]hCT binding were indistinguishable in MCF-7 and T47D cells. Yet, [125I]amylin binding was only recognized in MCF-7 cells, consistent with a distinct amylin receptor.

PGE2 binding sites and PG-stimulated cyclic AMP accumulation in rat isolated glomeruli and glomerular cultured cells

1983 ◽  
Vol 30 (2) ◽  
pp. 201-214 ◽  
Author(s):  
Gérard Friedlander ◽  
Dominique Chansel ◽  
Josée Sraer ◽  
Marcelle Bens ◽  
Raymond Ardaillou
Keyword(s):  
Cyclic Amp ◽  
Binding Sites ◽  
Cultured Cells ◽  
Isolated Glomeruli ◽  
Cyclic Amp Accumulation

scholarly journals The combination of carbon monoxide–haem with apoperoxidase

1969 ◽  
Vol 114 (4) ◽  
pp. 719-724 ◽  
Author(s):  
Charles Phelps ◽  
Eraldo Antonini
Keyword(s):  
Activation Energy ◽  
Carbon Monoxide ◽  
Rate Constant ◽  
Binding Sites ◽  
Order Process ◽  
First Order ◽  
Effects Of Ph ◽  
Kinetics Of

1. Static titrations reveal an exact stoicheiometry between various haem derivatives and apoperoxidase prepared from one isoenzyme of the horseradish enzyme. 2. Carbon monoxide–protohaem reacts rapidly with apoperoxidase and the kinetics can be accounted for by a mechanism already applied to the reaction of carbon monoxide–haem derivatives with apomyoglobin and apohaemoglobin. 3. According to this mechanism a complex is formed first whose combination and dissociation velocity constants are 5×108m−1sec.−1 and 103sec.−1 at pH9·1 and 20°. The complex is converted into carbon monoxide–haemoprotein in a first-order process with a rate constant of 235sec.−1 for peroxidase and 364sec.−1 for myoglobin at pH9·1 and 20°. 4. The effects of pH and temperature were examined. The activation energy for the process of complex-isomerization is about 13kcal./mole. 5. The similarity in the kinetics of the reactions of carbon monoxide–haem with apoperoxidase and with apomyoglobin suggests structural similarities at the haem-binding sites of the two proteins.

scholarly journals Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hajnalka Jankovics ◽  
Boglarka Kovacs ◽  
Andras Saftics ◽  
Tamas Gerecsei ◽  
Éva Tóth ◽  
...  
Keyword(s):  
Binding Sites ◽  
Kinetic Data ◽  
Specific Binding ◽  
Genetically Engineered ◽  
Binding Kinetics ◽  
Titration Calorimetry ◽  
Affinity Binding ◽  
Label Free ◽  
Low Protein ◽  
Kinetics Of

AbstractReliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the binding kinetics of Ni(II) ions to genetically modified flagellin layers, we demonstrate that: (1) Grating-Coupled Interferometry (GCI) is well suited to resolve the binding of ions, even at very low protein immobilization levels; (2) it supplies high quality kinetic data from which the number and strength of available binding sites can be determined, and (3) the rate constants of the binding events can also be obtained with high accuracy. Experiments were performed using a flagellin variant incorporating the C-terminal domain of the nickel-responsive transcription factor NikR. GCI results were compared to affinity data from titration calorimetry. We found that besides the low-affinity binding sites characterized by a micromolar dissociation constant (Kd), tetrameric FliC-NikRC molecules possess high-affinity binding sites with Kd values in the nanomolar range. GCI enabled us to obtain real-time kinetic data for the specific binding of an analyte with molar mass as low as 59 Da, even at signals lower than 1 pg/mm2.

scholarly journals Cannabidiol Selectively Binds to the Voltage-Gated Sodium Channel Nav1.4 in Its Slow-Inactivated State and Inhibits Sodium Current

Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1141
Author(s):  
Chiung-Wei Huang ◽  
Pi-Chen Lin ◽  
Jian-Lin Chen ◽  
Ming-Jen Lee
Keyword(s):  
Binding Sites ◽  
Sodium Current ◽  
Binding Kinetics ◽  
Muscle Membrane ◽  
Dependent Manner ◽  
Differential Inhibition ◽  
Fast Inactivation ◽  
Cell Recording ◽  
Domain I ◽  
Kinetics Of

Cannabidiol (CBD), one of the cannabinoids from the cannabis plant, can relieve the myotonia resulting from sodium channelopathy, which manifests as repetitive discharges of muscle membrane. We investigated the binding kinetics of CBD to Nav1.4 channels on the muscle membrane. The binding affinity of CBD to the channel was evaluated using whole-cell recording. The CDOCKER program was employed to model CBD docking onto the Nav1.4 channel to determine its binding sites. Our results revealed no differential inhibition of sodium current by CBD when the channels were in activation or fast inactivation status. However, differential inhibition was observed with a dose-dependent manner after a prolonged period of depolarization, leaving the channel in a slow-inactivated state. Moreover, CBD binds selectively to the slow-inactivated state with a significantly faster binding kinetics (>64,000 M−1 s−1) and a higher affinity (Kd of fast inactivation vs. slow-inactivation: >117.42 μM vs. 51.48 μM), compared to the fast inactivation state. Five proposed CBD binding sites in a bundle crossing region of the Nav1.4 channels pore was identified as Val793, Leu794, Phe797, and Cys759 in domain I/S6, and Ile1279 in domain II/S6. Our findings imply that CBD favorably binds to the Nav1.4 channel in its slow-inactivated state.

A quantitative assay for the cytoplasmic androgen receptor using [3H]dihydrotestosterone in the presence of NAD+-nucleosidase

1983 ◽  
Vol 102 (1) ◽  
pp. 153-160 ◽  
Author(s):  
Hans Moeller ◽  
Günter Oettling ◽  
Bernhard Fiederer ◽  
Gerd Brügmann
Keyword(s):  
Binding Sites ◽  
Androgen Receptors ◽  
Binding Capacity ◽  
Specific Binding ◽  
Linear Regression Analysis ◽  
Receptor Complex ◽  
Agar Gel ◽  
Progestin Receptors ◽  
Agar Gel Electrophoresis ◽  
Kinetics Of

Abstract. In prostatic cytosol DHT1 is metabolized to 5α-androstane-3α (or β), 17α-diols with a half life of 2 h even at 4°C. Thus, [3H]DHT appears to be a poor marker for a quantitative assessment of androgen receptors (AR). Methyltrienolone (R1881) seems to be advantageous as it is not metabolized. However, because of considerable binding to progestin receptors, assays using [3H]R1881 are not specific for AR in tissues containing progestin receptors. We, therefore, developed a specific assay for AR using [3H]DHT (14 nm) as marker, where metabolism of DHT is prevented by pre-incubation with NAD+-nucleosidase. The [3H]DHT-receptor complex is separated from free, SHBG-bound and unspecifically bound [3H]DHT by agar gel electrophoresis. The binding sites of high affinity and low capacity are characterized by suppression with unlabelled R1881 (2 μm) in a parallel assay. Under these conditions DHT and R1881 appear to have the same kinetics of association and dissociation. Weighted non-linear regression analysis of specific binding capacity at various ligand concentrations reveals that in rat prostatic cytosol the affinity of DHT (Kd = 0.405 ± 0.0839 nm) is significantly higher (P < 0.01) than that of R1881 (Kd = 1.25 ± 0.271 nm).

Brevity of the Ca2+ Microdomain and Active Zone Geometry Prevent Ca2+-Sensor Saturation for Neurotransmitter Release

2005 ◽  
Vol 94 (3) ◽  
pp. 1912-1919 ◽  
Author(s):  
Vahid Shahrezaei ◽  
Kerry R. Delaney
Keyword(s):  
Binding Sites ◽  
Neurotransmitter Release ◽  
Time Course ◽  
Receptor Occupancy ◽  
Channel Opening ◽  
Sensor Saturation ◽  
Highly Sensitive ◽  
Equilibrium Dissociation ◽  
Sensor Molecules ◽  
Kinetics Of

The brief time course of the calcium (Ca2+) channel opening combined with the molecular-level colocalization of Ca2+ channels and synaptic vesicles in presynaptic terminals predict sub-millisecond calcium concentration ([Ca2+]) transients of ≥100 μM in the immediate vicinity of the vesicle. This [Ca2+] is much higher than some of the recent estimates for the equilibrium dissociation constant of the Ca2+ sensor(s) that control neurotransmitter release, suggesting release should be close to saturation, yet it is well known that release is highly sensitive to changes in Ca2+ influx. We show that due to the brevity of the Ca2+ influx the binding kinetics of the Ca2+ sensor rather than its equilibrium affinity determine receptor occupancy. For physiologically relevant Ca2+ currents and forward Ca2+ binding rates, the effective affinity of the Ca2+ sensor can be several-fold lower than the equilibrium affinity. Using simple models, we show redundant copies of the binding sites increase effective affinity of the Ca2+ sensor for release. Our results predict that different levels of expression of Ca2+ binding sites could account for apparent differences in Ca2+ sensor affinities between synapses. Using Monte Carlo simulations of Ca2+ dynamics with nanometer resolution, we demonstrate that these kinetic constraints combined with vesicles acting as diffusion barriers can prevent saturation of the Ca2+-sensor(s) for neurotransmitter release. We further show the random positioning of the Ca2+-sensor molecules around the vesicle can result in the emergence of two distinct populations of the vesicles with low and high release probability. These considerations allow experimental evidence for the Ca2+ channel-vesicle colocalization to be reconciled with a high equilibrium affinity for the Ca2+ sensor of the release machinery.

scholarly journals Predominant expression of type-VI adenylate cyclase in C6-2B rat glioma cells may account for inhibition of cyclic AMP accumulation by calcium

1993 ◽  
Vol 293 (2) ◽  
pp. 325-328 ◽  
Author(s):  
M A Debernardi ◽  
R Munshi ◽  
M Yoshimura ◽  
D M Cooper ◽  
G Brooker
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Glioma Cells ◽  
Rat Glioma ◽  
Predominant Species ◽  
Cyclic Amp Accumulation ◽  
Substance K ◽  
Kinetics Of ◽  
Amp Inhibition ◽  
Predominant Expression

In C6-2B cells, agonist-stimulated cyclic AMP accumulation is inhibited when the cytosolic Ca2+ concentration is increased. We now demonstrate that in C6-2B cells: (i) the early kinetics of the cyclic AMP inhibition by substance K (t1/2 = 35 s) and thapsigargin (t1/2 = 1.6 min) closely mimic the kinetics of the cytosolic Ca2+ increase evoked by either agent (t1/2 = 25 s and 1.5 min respectively); (ii) the Ca2+ rise and cyclic AMP inhibition by substance K or thapsigargin are similarly affected in EGTA-containing medium; (iii) PCR detects type-III and type-VI adenylate cyclase cDNAs, and RNAase protection assays show that the mRNA for type-VI adenylate cyclase, an isoform inhibitable by submicromolar Ca2+ concentrations, is the predominant species, strongly suggesting that type-VI adenylate cyclase is probably the target molecule for Ca(2+)-mediated inhibition of cyclic AMP accumulation.

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