The topographical distributions and mobilities of the murine histocompatibility antigen H-2K(k) and of concanavalin A (Con A) binding sites have been studied on a murine lymphoma cell line. The spatial distribution of H-2K(k) antigens, the average distance between H-2K(k) antigens and Con A binding sites, and the separation of different determinants on the H-2K(k) antigen itself were determined by using fluorescence resonance energy-transfer measurements with a dual-laser flow sorter. From the lack of energy transfer between bound monoclonal anti-H-2K(k) antibodies conjugated with fluorescein (donor) and rhodamine (acceptor), we conclude that the H-2K(k) antigen exists without appreciable clustering on the cell surface. Substantial energy transfer between appropriately labeled Con A and antibodies bound to the H-2K(k) antigen shows that the two populations are interspersed, Donor/acceptor pairs of monoclonal antibodies binding to different determinants on the same H-2K(k) antigen exhibited a degree of energy transfer indicative of a mean separation of 8.6 nm between the sites. Time-resolved phosphorescence anisotropy measurements with anti-H-2K(k) antibodies labeled with eosin or erythrosin yielded rotational mobility information for the antigen-antibody complexes on the cell membrane. The rotational correlation time of 10-20 μs and the finite residual anisotropy are compatible with an uniaxial mode of rotation of monomeric antigen around its transmembrane portion and, thus, provide additional evidence for an unclustered distribution. Capping by rabbit anti-mouse IgG immobilized the antigen-antibody complex. Fluorescence recovery after photobleaching was used to calculate an apparent lateral diffusion coefficient of 5 ± 3 x 10-10 cm2.s-1 for the H-2K(k) antigen labeled with fluoresceinated IgG or its corresponding Fab fragment.
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Issue number||19 I|
|Publication status||Published - Jan 1 1983|
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