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.

Studies of the uptake of macromolecules by cells. I. Uptake of proteins by cells of the Ehrlich–Lettré ascites carcinoma

1968 ◽  
Vol 46 (12) ◽  
pp. 1443-1450 ◽  
Author(s):  
Y. C. Choi ◽  
E. R. M. Kay
Keyword(s):  
Nucleic Acid ◽  
Cell Membrane ◽  
Binding Sites ◽  
Specific Binding ◽  
Protein Uptake ◽  
Specific Binding Sites ◽  
Model Protein ◽  
Uptake Process ◽  
Kinetics Of ◽  
Protein Treatment

The uptake of protein by cells of the Ehrlich–Lettré ascites carcinoma was characterized kinetically by using hemoglobin as a model protein. An attempt was made to show that the process is not an artefact due to nonspecific adsorption of protein to the cell membrane. The kinetics of the uptake process suggested that an interaction exists between the exogenous protein and specific binding sites on the membrane. Acetylation of hemoglobin enhanced the rate of uptake of this protein. Treatment of cells with neuraminidase, phospholipase A, and Pronase resulted in an inhibition of protein uptake. The experimental evidence for the uptake of hemoglobin was supported by evidence that L-serine-U-14C-labelled hemoglobin is transported into the cytoplasm and utilized subsequently, resulting in labelling of the nucleic acid nucleotides.

Cholecystokinin downregulates receptors for vasoactive intestinal peptide and secretin in rat pancreatic acini

1990 ◽  
Vol 258 (3) ◽  
pp. G395-G403
Author(s):  
S. Katsushima ◽  
H. Adachi ◽  
T. Honda ◽  
S. Sato ◽  
T. Kusui ◽  
...  
Keyword(s):  
Vasoactive Intestinal Peptide ◽  
Binding Sites ◽  
Specific Binding ◽  
Amylase Secretion ◽  
Affinity Binding ◽  
Pancreatic Acini ◽  
Cck Receptors ◽  
Cyclic Monophosphate ◽  
Apparent Decrease ◽  
Specific Antagonist

We examined the effect of cholecystokinin (CCK) on the receptors for vasoactive intestinal peptide (VIP) and secretin in rat pancreatic acini. CCK decreased the specific binding of 125I-VIP and 125I-secretin by 42 and 51%, respectively. This CCK-induced inhibition was caused by an apparent decrease in the capacity of high-affinity binding sites of VIP and secretin receptors. CR 1409, a specific antagonist of CCK, abolished CCK-induced binding inhibition, whereas 12-O-tetradecanoylphorbol-13-acetate, A23187, and cycloheximide did not affect the binding of the radioligands. Both N2,O2-dibutyryl guanosine 3',5'-cyclic monophosphate (Bt2cGMP) and nitroprusside inhibited the specific binding of 125I-VIP. This inhibition, however, was because of an apparent decrease in the capacity of low-affinity binding sites on VIP receptors. CCK-induced downregulation of VIP and secretin receptors was associated with the diminished acinar response to VIP or secretin-induced adenosine 3',5'-cyclic monophosphate accumulation and amylase secretion, whereas neither Bt2cGMP nor nitroprusside affected VIP-induced amylase secretion. Data suggest that CCK-induced downregulation is mediated by the initial interaction of CCK with CCK receptors followed by some postreceptor process, which appears unrelated to protein kinase C, calcium mobilization, decrease in protein synthesis, or cellular cGMP increases. This downregulation, at least in part, accounts for CCK-induced restricted stimulation of amylase secretion by VIP and secretin.

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 Insights into Hydrophobicity and the Chaperone-like Function of αA- and αB-crystallins

2005 ◽  
Vol 280 (23) ◽  
pp. 21726-21730 ◽  
Author(s):  
M. Satish Kumar ◽  
Mili Kapoor ◽  
Sharmistha Sinha ◽  
G. Bhanuprakash Reddy
Keyword(s):  
Binding Sites ◽  
Chaperone Activity ◽  
Titration Calorimetry ◽  
Affinity Binding ◽  
Hydrophobic Surfaces ◽  
Ans Binding ◽  
High Affinity Binding ◽  
Chaperone Function ◽  
High Affinity ◽  
Αb Crystallin

α-Crystallin, composed of two subunits, αA and αB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of α-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of αA- and αB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of αA- and αB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with αB showing a higher number of sites than αA at 30 °C. In agreement with the higher number of hydrophobic sites, αB-crystallin demonstrated higher chaperone activity than αA at this temperature. Thermodynamic analysis of ANS binding to αA- and αB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for αA and αB at 15 °C, αA was more potent than αB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of αA and αB for ANS upon preheating, there was an increase in the number of low affinity sites of αA and αB. Preheated αA, in contrast to αB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of α-crystallins, it does not quantitatively correlate with the chaperone function of α-crystallins.

Analysis of PTHRP binding and signal transduction mechanisms in benign and malignant squamous cells

1992 ◽  
Vol 262 (5) ◽  
pp. E599-E607 ◽  
Author(s):  
J. J. Orloff ◽  
M. B. Ganz ◽  
A. E. Ribaudo ◽  
W. J. Burtis ◽  
M. Reiss ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Binding Sites ◽  
Specific Binding ◽  
High Capacity ◽  
Squamous Carcinoma ◽  
Epidermal Keratinocytes ◽  
Affinity Binding ◽  
Human Epidermal Keratinocytes ◽  
Squamous Carcinoma Cells ◽  
Parathyroid Hormone Related Protein

We have explored a potential autocrine role for parathyroid hormone-related protein (PTHRP) in malignant squamous carcinoma cells (SqCC) and their nonmalignant counterpart, human epidermal keratinocytes (HK). Specific binding of Tyr36 human PTHRP-(1-36)NH2 (125I-[Tyr36]hPTHRP-(1-36)NH2) was identified in 75% of unselected SqCC lines. In contrast, no binding was detected on the mouse keratinocyte line BALB-MK or on five different HK lines. Although each SqCC and keratinocyte line secreted immunoreactive PTHRP into its medium, there was no correlation between PTHRP concentration and number of binding sites. Inhibition of binding by [Tyr36]hPTHRP-(1-36)NH2 yielded half-maximal inhibitory concentration values of approximately 100 nM in all SqCC lines. Affinity cross-linking of SqCC cells revealed 98- and 70-kDa binding proteins with similar affinity (approximately 100 nM). Exposure of fura-2-loaded SqCC cells to PTHRP and PTH resulted in equivalent, dose-dependent transient increases in intracellular calcium [half-maximal effective concentration (EC50) = 0.08 nM]. PTHRP also increased intracellular calcium in HK (EC50 = 0.05 nM). No adenosine 3',5'-cyclic monophosphate (cAMP) response to PTHRP or PTH was elicited in either SqCC or HK, despite brisk isoproterenol responses in both. We conclude that high-capacity low-affinity binding sites for PTHRP are detectable in the majority of SqCC lines but not in HK. These low-affinity binding sites are unlikely to represent receptors. The sensitive intracellular calcium response suggests the additional presence of high-affinity receptors on SqCC as well as on HK. However, the failure of PTHRP or PTH to stimulate cAMP production in otherwise cyclase-competent cells suggests that these are not classical PTH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

scholarly journals Discrimination between G/C Binding Sites by Olivomycin A Is Determined by Kinetics of the Drug-DNA Interaction

2020 ◽  
Vol 21 (15) ◽  
pp. 5299
Author(s):  
Artemy D. Beniaminov ◽  
Galina V. Chashchina ◽  
Mikhail A. Livshits ◽  
Olga I. Kechko ◽  
Vladimir A. Mitkevich ◽  
...  
Keyword(s):  
Binding Sites ◽  
Dna Interaction ◽  
In Vitro Transcription ◽  
Dissociation Rate ◽  
Titration Calorimetry ◽  
Drug Candidates ◽  
Important Approach ◽  
Functional Relevance ◽  
Kinetics Of

Olivomycin A (OA) exerts its cytotoxic potency due to binding to the minor groove of the G/C-rich DNA and interfering with replication and transcription. Screening of the complete set of tetranucleotide G/C sites by electrophoretic mobility gel shift assay (EMSA) revealed that the sites containing central GC or GG dinucleotides were able to bind OA, whereas the sites with the central CG dinucleotide were not. However, studies of equilibrium OA binding in solution by fluorescence, circular dichroism and isothermal titration calorimetry failed to confirm the sequence preference of OA, indicating instead a similar type of complex and comparable affinity of OA to all G/C binding sites. This discrepancy was resolved by kinetics analysis of the drug–DNA interaction: the dissociation rate significantly differed between SGCS, SGGS and SCGS sites (S stands for G or C), thereby explaining the disintegration of the complexes during EMSA. The functional relevance of the revealed differential kinetics of OA–DNA interaction was demonstrated in an in vitro transcription assay. These findings emphasize the crucial role of kinetics in the mechanism of OA action and provide an important approach to the screening of new drug candidates.

scholarly journals Characterization of the Binding of Adenosine Diphosphate to Human Platelet Membranes

1977 ◽  
Author(s):  
C. Legrand ◽  
B. Bauvois ◽  
J. P. Caen
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Adenosine Diphosphate ◽  
Affinity Constant ◽  
Membrane Bound ◽  
High Concentrations ◽  
Kinetics Of

ADP-mediated platelet aggregation is a routinely employed test but its mechanism is poorly understood. The aim of this study was to compare the binding of ADP to plasma membranes isolated from normal platelets and thrombasthenic platelets (which do not aggregate with ADP). Binding of ADP to isolated membranes was assayed by incubation with 14C-ADP followed by Mill i pore filtration. In standard conditions, 14C-ADP was not transformed and non specific binding represented lessthan 3 % of the total binding. Using 1 μM 14C-ADP, the binding has been shown to be a rapid (t 1/2 = 2 mn 30 sec), saturable and reversible phenomenon at 37° C. The existence of a major population of binding sites, with an affinity constant Ka = 0.43 (+ 0.1) χ 106M-1, has been demonstrated. The kinetics of the binding was normal with membranes Tsolated from the platelets of 4 thrombasthenic patients and the affinity constant, when determined, was in the normal range. Dissociation of the membrane-bound 14C-ADP occurred rapidly at 37° C (t l/2c≃3mn) when samples were diluted enough (dilution 1 : 100 was currently employed) to avoid rebinding of the radioligand. Accelerated dissociation (t 1/2 ≃ 1 mn) was observed when the dilution was performed in the presence of an excess of unlabeled ADP, suggesting the existence of negatively cooperative site-site interactions among the ADP binding sites. This effect was only observed at high concentrations of ADP (> 10–5M) and its eventual role in vivo remains to be established. Two thrombasthenic membrane preparations studied in the same way dissociated as did the control membranes.

scholarly journals Structure of a highly acidic β-lactamase from the moderate halophileChromohalobactersp. 560 and the discovery of a Cs+-selective binding site

2015 ◽  
Vol 71 (3) ◽  
pp. 541-554 ◽  
Author(s):  
Shigeki Arai ◽  
Yasushi Yonezawa ◽  
Nobuo Okazaki ◽  
Fumiko Matsumoto ◽  
Chie Shibazaki ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Nuclear Power ◽  
Specific Binding ◽  
Radioactive Isotopes ◽  
Titration Calorimetry ◽  
X Ray ◽  
X Ray Crystallography ◽  
Specific Binding Sites ◽  
Enzymatic Function

Environmentally friendly absorbents are needed for Sr2+and Cs+, as the removal of the radioactive Sr2+and Cs+that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs+or Sr2+. The crystal structure of a halophilic β-lactamase fromChromohalobactersp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space groupP31using X-ray crystallography. Moreover, the locations of bound Sr2+and Cs+ions were identified by anomalous X-ray diffraction. The location of one Cs+-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na+(90 mMNa+/10 mMCs+). From an activity assay using isothermal titration calorimetry, the bound Sr2+and Cs+ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs+-binding site provides important information that is useful for the design of artificial Cs+-binding sites that may be useful in the bioremediation of radioactive isotopes.

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.

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