Binding of the soluble, truncated form of an Fc receptor (mouse Fc gamma RII) to membrane-bound IgG as measured by total internal reflection fluorescence microscopy.
Total internal reflection fluorescence microscopy has been used to investigate the binding of the soluble extracellular domain of mouse Fc gamma RII (sFc gamma RII) to an anti-trinitrophenyl monoclonal mouse IgG2b (GK14.1) specifically bound to substrate-supported planar membranes composed of dipalmitoylphosphatidylcholine (DPPC) and trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine (TNP-cap-DPPE). The equilibrium dissociation constants for sFc gamma RII at GK14.1-coated TNP-cap-DPPE/DPPC planar membranes containing 0.5-25 mol% TNP-cap-DPPE were approximately 1 microM. Total internal reflection with fluorescence photobleaching recovery was used to examine the dissociation kinetics. The fluorescence recovery curves were better described as a sum of two exponentials rather than by one exponential; the rates and fractional recoveries were approximately 1 s-1 (65%) and approximately 0.1 s-1 (35%). The similarity between the values of these equilibrium and kinetic parameters to those previously measured for the binding of IgG in solution to intact mouse Fc gamma RII reconstituted into planar membranes suggests that conformational changes which may occur when IgG is constrained to a membrane surface do not significantly affect the equilibrium or kinetics of IgG-mouse Fc gamma RII binding. The stoichiometry of sFc gamma RII-GK14.1 binding was 1:4, indicating that a significant fraction of the membrane-bound antibodies were not accessible for receptor binding. Possible mechanisms that might underlay the observed heterogeneity in sFc gamma RII-IgG binding kinetics are discussed.
Gesty-Palmer
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Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290, USA.
Thompson
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D015060:1,2-Dipalmitoylphosphatidylcholine; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001665:Binding Sites; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D008567:Membranes, Artificial; D051379:Mice; D008856:Microscopy, Fluorescence; D010714:Phosphatidylethanolamines; D011485:Protein Binding; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D017452:Receptors, IgG / Q000276:immunology / Q000378:metabolism*; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D008567:Membranes, Artificial; D010714:Phosphatidylethanolamines; D011947:Receptors, Antigen, B-Cell; D017452:Receptors, IgG; D015060:1,2-Dipalmitoylphosphatidylcholine; C033368:N-(2,4,6-trinitrophenyl-6-N-aminocaproyl)-1,2-dipalmitoylphosphatidylethanolamine