High-affinity fluorescent peptide binding to I-Ad in lipid membranes.
Membranes isolated from antigen-pulsed, antigen-presenting cells were extensively dialyzed and shown to retain the ability to present antigen to an I-Ad-restricted, antigen-specific T cell, 3DO-54.8. This ability to retain antigen on the membrane was duplicated in lysates of antigen-presenting cells as well as with pure I-A molecules in phospholipid vesicles. Measurement of the concentration of surface-associated fluorescent peptide on planar membranes prepared from antigen-pulsed phospholipid vesicles showed that about 1 peptide molecule was retained per 100 I-A molecules. We calculate that about 1000 I-A-associated peptide molecules are sufficient to stimulate the response of a single 3DO-54.8 cell. The association of fluorescent peptide with the surface depended on the presence of I-A and was blocked by unlabeled ovalbumin peptide or by a digest of hen egg lysozyme, added at the time of the pulse, but not after pulsing.
D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology*; D002462:Cell Membrane / Q000276:immunology; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D008081:Liposomes; D008211:Lymphocyte Cooperation; D051379:Mice; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D010047:Ovalbumin / Q000276:immunology; D011485:Protein Binding; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*; D013861:Thiocyanates
D005452:Fluoresceins; D000949:Histocompatibility Antigens Class II; D008081:Liposomes; D009842:Oligopeptides; D013861:Thiocyanates; D010047:Ovalbumin; D016650:Fluorescein-5-isothiocyanate