Mechanism of the regulation of type IB phosphoinositide 3OH-kinase byG-protein βγ subunits

Type IB phosphoinositide 3OH-kinase (PI3K) is activated by G-protein βγ subunits (Gβγs). The enzyme is soluble and largely cytosolic in vivo. Its substrate, PtdIns(4,5)P2, and the Gβγs are localized at the plasma membrane. We have addressed the mechanism by which Gβγs regulate the PI3K using an in vitro approach. We used sedimentation assays and surface plasmon resonance to determine association of type IB PI3K with lipid monolayers and vesicles of varying compositions, some of which had Gβγs incorporated. Association and dissociation rate constants were determined. Our results indicated that in an assay situation in vitro the majority of PI3K will be associated with lipid vesicles, irrespective of the presence or absence of Gβγs. In line with this, a constitutively active membrane-targeted PI3K construct could still be activated substantially by Gβγs in vitro. We conclude that Gβγs activate type IB PI3K by a mechanism other than translocation to the plasma membrane.

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Quick Evaluation of Kinase Inhibitors by Surface Plasmon Resonance Using Single-Site Specifically Biotinylated Kinases

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057113506051 ◽

2013 ◽

Vol 19 (3) ◽

pp. 453-461 ◽

Cited By ~ 7

Author(s):

Daisuke Kitagawa ◽

Masaki Gouda ◽

Yasuyuki Kirii

Keyword(s):

Surface Plasmon Resonance ◽

Kinetic Parameters ◽

Surface Plasmon ◽

Plasmon Resonance ◽

Kinase Inhibitors ◽

Growth Factor Receptor ◽

Biochemical Properties ◽

Dissociation Rate ◽

Single Site ◽

Dissociation Rate Constants

In evaluating kinase inhibitors, kinetic parameters such as association/dissociation rate constants are valuable information, as are equilibrium parameters KD and IC50 values. Surface plasmon resonance (SPR) is a powerful technique to investigate these parameters. However, results are often complicated because of impaired conformations by inappropriate conditions required for protein immobilization and/or heterogeneity of the orientation of immobilization. In addition, conventional SPR experiments are generally time-consuming. Here we introduce the use of single-site specifically biotinylated kinases combined with a multichannel SPR device to improve such problems. Kinetic parameters of four compounds—staurosporine, dasatinib, sunitinib, and lapatinib—against six kinases were determined by the ProteOn XPR36 system. The very slow off-rate of lapatinib from the epidermal growth factor receptor and dasatinib from Bruton’s tyrosine kinase and colony stimulating factor 1 receptor (CSF1R) were confirmed. Furthermore, IC50 values were determined by an activity-based assay. Evaluating both physicochemical and biochemical properties would help to understand the detailed character of the compound.

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Surface plasmon resonance: a useful technique for cell biologists to characterize biomolecular interactions

Molecular Biology of the Cell ◽

10.1091/mbc.e12-10-0713 ◽

2013 ◽

Vol 24 (7) ◽

pp. 883-886 ◽

Cited By ~ 44

Author(s):

Robert V. Stahelin

Keyword(s):

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Protein Interactions ◽

Plasmon Resonance ◽

Dissociation Rate ◽

Biomolecular Interactions ◽

Protein Protein Interactions ◽

Interaction Kinetics ◽

Lipid Protein Interactions ◽

Dissociation Rate Constants

Surface plasmon resonance (SPR) is a powerful technique for monitoring the affinity and selectivity of biomolecular interactions. SPR allows for analysis of association and dissociation rate constants and modeling of biomolecular interaction kinetics, as well as for equilibrium binding analysis and ligand specificity studies. SPR has received much use and improved precision in classifying protein–protein interactions, as well as in studying small-molecule ligand binding to receptors; however, lipid–protein interactions have been underserved in this regard. With the field of lipids perhaps the next frontier in cellular research, SPR is a highly advantageous technique for cell biologists, as newly identified proteins that associate with cellular membranes can be screened rapidly and robustly for lipid specificity and membrane affinity. This technical perspective discusses the conditions needed to achieve success with lipid–protein interactions and highlights the unique lipid–protein interaction mechanisms that have been elucidated using SPR. It is intended to provide the reader a framework for quantitative and confident conclusions from SPR analysis of lipid–protein interactions.

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An in vitro assay based on surface plasmon resonance to predict the in vivo circulation kinetics of liposomes

Journal of Controlled Release ◽

10.1016/j.jconrel.2011.07.023 ◽

2011 ◽

Vol 156 (3) ◽

pp. 307-314 ◽

Cited By ~ 24

Author(s):

B.J. Crielaard ◽

A. Yousefi ◽

J.P. Schillemans ◽

C. Vermehren ◽

K. Buyens ◽

...

Keyword(s):

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Plasmon Resonance ◽

In Vitro Assay ◽

Vitro Assay ◽

Kinetics Of

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Surface plasmon resonance unveils important pitfalls of enzyme-linked immunoassay for the detection of anti-infliximab antibodies in patients’ sera

Scientific Reports ◽

10.1038/s41598-021-94431-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marten Beeg ◽

Cesare Burti ◽

Eleonora Allocati ◽

Clorinda Ciafardini ◽

Rita Banzi ◽

...

Keyword(s):

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Plasmon Resonance ◽

Dissociation Rate ◽

Therapeutic Antibodies ◽

Serum Concentrations ◽

High Affinity ◽

Bowel Diseases ◽

Inflammatory Bowel ◽

Dissociation Rate Constants

AbstractMeasurements of serum concentrations of therapeutic antibodies and anti-drug antibodies (ADA) can support clinical decisions for the management of non-responders, optimizing the therapy. In the present study we compared the results obtained by classical ELISA and a recently proposed surface plasmon resonance (SPR)-based immunoassay, in 76 patients receiving infliximab for inflammatory bowel diseases. The two methods indicated very similar serum concentrations of the drug, but there were striking differences as regards ADA. All the sera showing ADA by ELISA (14) also showed ADA by SPR, but the absolute amounts were different, being 7–490 times higher with SPR, with no correlation. Eight patients showed ADA only with SPR, and these ADA had significantly faster dissociation rate constants than those detectable by both SPR and ELISA. The underestimation, or the lack of detection, of ADA by ELISA is likely to reflect the long incubation steps which favor dissociation of the patient’s low-affinity ADA, while the commercial, high-affinity anti-infliximab antibodies used for the calibration curve do not dissociate. This problem is less important with SPR, which monitors binding in real time. The possibility offered by SPR to detect ADA in patients otherwise considered ADA-negative by ELISA could have important implications for clinicians.

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Surface modification of gold nanoparticles by cetirizine through surface plasmon resonance and preliminary study of the in vitro cellular cytotoxicity

Journal of Molecular Liquids ◽

10.1016/j.molliq.2021.115542 ◽

2021 ◽

Vol 330 ◽

pp. 115542

Author(s):

Lifeng Liu ◽

Ehsan Koushki ◽

Reza Tayebee

Keyword(s):

Gold Nanoparticles ◽

Surface Modification ◽

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Plasmon Resonance ◽

Cellular Cytotoxicity ◽

Preliminary Study

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A New Surface Plasmon Resonance Assay for In Vitro Screening of Mannose-Binding Lectin Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057116637563 ◽

2016 ◽

Vol 21 (7) ◽

pp. 749-757 ◽

Cited By ~ 4

Author(s):

Matteo Stravalaci ◽

Daiana De Blasio ◽

Franca Orsini ◽

Carlo Perego ◽

Alessandro Palmioli ◽

...

Keyword(s):

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Plasmon Resonance ◽

Ischemia Reperfusion ◽

Mannose Binding Lectin ◽

Lectin Pathway ◽

In Vitro Screening ◽

Mannose Binding ◽

Binding Lectin

Mannose-binding lectin (MBL) is a circulating protein that acts as a soluble pattern recognition molecule of the innate immunity. It binds to carbohydrate patterns on the surface of pathogens or of altered self-cells, with activation of the lectin pathway of the complement system. Recent evidence indicates that MBL contributes to the pathophysiology of ischemia-reperfusion injury and other conditions. Thus, MBL inhibitors offer promising therapeutic strategies, since they prevent the interaction of MBL with its target sugar arrays. We developed and characterized a novel assay based on surface plasmon resonance for in vitro screening of these compounds, which may be useful before the more expensive and time-consuming in vivo studies. The assay measures the inhibitor’s ability to interfere with the binding of murine MBL-A or MBL-C, or of human recombinant MBL, to mannose residues immobilized on the sensor chip surface. We have applied the assay to measure the IC50 of synthetic glycodendrimers, two of them with neuroprotective properties in animal models of MBL-mediated injuries.

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Dynamics of Dental Pellicle Formation - In Vitro Analysis of Time Dependant Binding Behavior by Surface Plasmon Resonance and the Influence of Oral Therapeutics

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.415.77 ◽

2009 ◽

Vol 415 ◽

pp. 77-80

Author(s):

Doru Vornicescu ◽

Katerina Solanska ◽

Ioana Demetrescu ◽

Matthias Frentzen ◽

Michael Keusgen

Keyword(s):

Surface Plasmon Resonance ◽

Real Time ◽

Surface Plasmon ◽

Plasmon Resonance ◽

Artificial Saliva ◽

Human Saliva ◽

Forming Process ◽

Pellicle Formation ◽

Binding Behavior

: The pellicle on oral surfaces represents a central interface for the formation of biofilms. Among other things it causes the first adsorption of bacteria. The dynamics of pellicle formation, on tooth surfaces and the influence of oral therapeutics on the pellicle structure are fairly unknown. With the method of surface plasmon resonance (SPR), the formation of salivary pellicle structures on hydroxylapatite (HAP) surfaces covering a very thin (~50nm) layer gold on a glass prism was recorded in real time without labeling or destruction. As pellicle forming substrates natural pooled human saliva (NS) and artificial saliva (AS) were used. To simulate the influence of therapeutic additives on the dynamic of the pellicle forming process, a chlorhexidine preparate (Chlorhexamed Fluid® CHX) on two different concentrations was selected. The binding behavior of a NS and a preparation in terms of an AS were compared. The layer was largely stable against rinsing with buffer. The application of CHX preparations in two different concentrations as an example of an oral therapeutic additive revealed a complex dynamic of adsorption. CHX did not lead to any visible destruction of the pellicle. The introduced method is an excellent tool to illustrate the dynamic effects of pellicle formation or pellicle reorganization by measuring the increase or decrease of the SPR signal in real time.

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