The use of nonhomologous Scatchard analysis in the evaluation of ligand–protein interactions

Zoelen, E.J.J. van; Kramer, R.H.; Moerkerk, H.T.B. van; Veerkamp, J.H.

doi:10.1016/s0165-6147(98)01250-4

Cited by 12 publications

(8 citation statements)

References 17 publications

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“…Similarly, the dose‐response curves of the competition assay were fitted, while the K i for the competitors were obtained not only from the one‐site competition model but also from the two‐site model if applicable. A Scatchard plot was applied to the competitors using a specific data transformation for the nonhomologous competition . Because we used the total concentration of the competitors in substituting the free one in the analysis, the binding data were excluded at low concentrations (≤1 nM) due to possible depletion of the competitors.…”

Section: Methodsmentioning

confidence: 99%

“…A Scatchard plot was applied to the competitors using a specific data transformation for the nonhomologous competition. 45 Because we used the total concentration of the competitors in substituting the free one in the analysis, the binding data were excluded at low concentrations (≤1 nM) due to possible depletion of the competitors. Kinetics in association and dissociation analyses was fitted by GraphPad Prism 8 according to one-phase exponential equations or to two-phase equations if applicable.…”

Section: Data and Statistical Analysismentioning

confidence: 99%

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Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT

Song

Shen

et al. 2020

FASEB j.

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The glucagon‐like peptide‐1 receptor (GLP‐1R), a family B G‐protein coupled receptor (GPCR), regulates the insulin secretion following stimulation by ligands. The transmembrane domain (TM) mediates GLP‐1R homodimerization, which modulates its ligand binding and signaling. We investigated the possible involvement of the N‐terminal extracellular domain (NTD) in dimerization/oligomerization and dimer‐associated ligand binding by NanoLuc Binary Technology (NanoBiT). With improved NanoBiT detection using a decreasing substrate concentration, the negative cooperativity of ligand binding to the NTD was confirmed by accelerated dissociation and Scatchard analysis. The dimerization/oligomerization of the isolated NTD was observed by NanoBiT and validated by analytical ultracentrifugation, deriving the comparable dimerization affinity (~105 M−1). The NTD was also involved in the dimerization/oligomerization of the full‐length GLP‐1R with mutated TM4 at the cellular level. In an analysis of the parameters of the NTD binding, the Kd for the probe GLP‐1 (7‐36, A8G) was similar (6‐8 μM) in both the 1:1 binding model and the receptor dimerization model. Compared with GLP‐1 and dulaglutide, exenatide showed two‐site binding with Ki values of 1.4 pM and 8.7 nM. Our study indicates the involvement of NTD in the GLP‐1R dimerization/oligomerization and suggests that further investigations on the role in other family B GPCRs are needed.

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Section: Methodsmentioning

confidence: 99%

Section: Data and Statistical Analysismentioning

confidence: 99%

Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT

Song

Shen

et al. 2020

FASEB j.

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“…Results were analyzed statistically using ANOVA (37). Scatchard analyses of ligand binding data were conducted as previously outlined (38). Comparisons between treatment means, where applicable, were achieved with the student‐Neuman‐Keul's test.…”

Section: Methodsmentioning

confidence: 99%

β‐Adrenergic Agonist Hyperplastic Effect Is Associated with Increased Fibronectin Gene Expression and Not Mitogen‐Activated Protein Kinase Modulation in C₂C₁₂ Cells

Izevbigie

Bergen

2000

Proc Soc Exp Biol Med

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Abstract. β‐Adrenergic agonists (β‐AA) enhance protein accretion in skeletal muscles. This stimulation is characterized by increased protein synthesis, increased expression of myofibrillar protein genes and a depression in protein degradation in animals, and increased proliferation and DNA synthesis in muscle cells in vitro. The mechanism or signal path in muscle whereby β‐AA would elicit these physiological effects upon binding to the G protein–coupled β‐adrenergic receptor (β‐AR) is unclear. C2C12 myoblasts were used to determine β‐AR ligand binding characteristics, cyclic AMP synthesis in response to isoproterenol (ISO) stimulation, and effects of ISO on DNA synthesis, mitogen activated protein kinase (MAPK), and fibronectin (FN) gene expression. Results showed that C2C12 cells possess β‐AR which are specific, saturable, and of high affinity (Kd = 0.2 nM). Forskolin and ISO stimulated cAMP production by ≈ 20‐fold (P < 0.001) and 17‐fold (P < 0.001), respectively. ISO and the cAMP analog, 8‐bromo‐cAMP (8‐BC) stimulated DNA synthesis in proliferating cells by 150% (P < 0.05) and 200% (P < 0.01), respectively, without modulating MAPK activity, whereas addition of fetal bovine serum to culture resulted in a 500% increase (P < 0.01) in DNA synthesis and MAPK activation. DNA synthesis in C2C12 cells treated with ISO, 8‐BC, or FBS was abolished in the presence of 25 μM PD098059, an MAPK‐kinase inhibitor, suggesting that an MAPK‐dependent pathway is likely involved in C2C12 proliferation. During cAMP elevating agent stimulation, basal MAPK activity may be sufficient, in the presence of other putative signaling molecules, to support proliferation in these cells. ISO or 8‐BC treatment increased FN mRNA by three‐ and seven‐fold, respectively, in growing C2C12 cells implying a connection between increased DNA synthesis and FN gene expression.

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“…The molecular recognition properties of the obtained polymers were estimated according to Scatchard analysis [24]. Figure 6 showed the binding isotherms for CLB on CLB-MIMSP and NIPs.…”

Section: Determination Of Binding Parametersmentioning

confidence: 99%

Preparation of Monolithic Imprinted Stationary Phase for Clenbuterol by In Situ Polymerization and Application in Biological Samples Pretreatment

Luo

Huang

et al. 2011

Chromatographia

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A clenbuterol (CLB) molecule-imprinted monolithic stationary phase (MIMSP) with specific recognition for CLB and some other b 2 -adrenergic receptor agonists was prepared by in situ polymerization technique utilizing methacrylic acid as a functional monomer, ethylene glycol dimethacrylate (EDMA) as a cross-linking reagent, and low polar solvents (toluene and dodecanol) as porogenic solvents. The optimal polymerization conditions were as follows: the molar ratio of template:monomer:initiator was 5:20:1, EDMA was 85% (v/v) in the total volume of monomer and EDMA, and toluene was 18% (v/v) in the total mixed porogen. The selectivity of the stationary phase for CLB and other b 2 -adrenergic receptor agonists was evaluated by high performance liquid chromatography. Scatchard analysis was employed to explore the recognition mechanism. Then the CLB-MIMSP was used as a solid phase extraction (SPE) stationary phase for concentration and purification of CLB from pig liver samples. The results showed that the obtained CLB-MIMSP possessed high selectivity towards CLB and moderate selectivity towards some other b 2 -adrenergic receptor agonists with characteristics of easy-made. The limit of detection was 10 ng g -1 , and recoveries of CLB were 99.16-113.06% with RSD 4.55-11.81% for the spiked pig liver samples. The CLB-MIMSP could be a promising SPE absorbent in CLB biological sample pretreatment.

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The use of nonhomologous Scatchard analysis in the evaluation of ligand–protein interactions

Cited by 12 publications

References 17 publications

Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT

Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT

β‐Adrenergic Agonist Hyperplastic Effect Is Associated with Increased Fibronectin Gene Expression and Not Mitogen‐Activated Protein Kinase Modulation in C₂C₁₂ Cells

Preparation of Monolithic Imprinted Stationary Phase for Clenbuterol by In Situ Polymerization and Application in Biological Samples Pretreatment

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The use of nonhomologous Scatchard analysis in the evaluation of ligand–protein interactions

Cited by 12 publications

References 17 publications

Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT

Dimerization/oligomerization of the extracellular domain of the GLP‐1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT

β‐Adrenergic Agonist Hyperplastic Effect Is Associated with Increased Fibronectin Gene Expression and Not Mitogen‐Activated Protein Kinase Modulation in C2C12 Cells

Preparation of Monolithic Imprinted Stationary Phase for Clenbuterol by In Situ Polymerization and Application in Biological Samples Pretreatment

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Product

Resources

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β‐Adrenergic Agonist Hyperplastic Effect Is Associated with Increased Fibronectin Gene Expression and Not Mitogen‐Activated Protein Kinase Modulation in C₂C₁₂ Cells