Structural mapping of Fc receptor bound immunoglobulin E: proximity to the membrane surface of the antibody combining site and another site in the Fab segments.
Resonance energy-transfer methods have been used to investigate the structure of immunoglobulin E (IgE) bound to its high-affinity receptor on plasma membrane vesicles derived from rat basophilic leukemia cells. The structural mapping of receptor-bound IgE was initiated in an earlier study [Holowka, D., & Baird, B. (1983) Biochemistry 22, 3475], and it is based on measuring the minimal distance from IgE sites that are selectively labeled with donor probes to a plane of amphipathic acceptors at the membrane surface. This paper describes the use of monoclonal IgE specific for 5-(dimethylamino)naphthalene-1-sulfonyl (DNS) to place a donor probe, DNS-L-Lys, in the antibody combining sites. The distance from these sites to the membrane surface was determined to be greater than 100 A with two different amphipathic acceptor probes. Another site in the Fab segments of monoclonal IgE (anti-dinitrophenyl) could be labeled selectively with N-[4-[7-(diethylamino)-4-methylcoumarin-3-yl]phenyl]maleimide (CPM) in the absence of reducing agents [CPM(-)], and the reaction could not be blocked by prereaction with N-ethylmaleimide. The pattern of CPM(-)-labeled proteolytic fragments and the lack of fluorescence quenching by (trinitrophenyl)lysine in the antibody combining sites suggested the CPM(-)-labeled site to be in the C epsilon 1 domain of IgE. The distance between this site on receptor-bound IgE and the membrane surface was determined to be 75-87 A with two different amphipathic acceptors.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000911:Antibodies, Monoclonal; D001491:Basophils / Q000201:enzymology; D002462:Cell Membrane / Q000276:immunology; D004735:Energy Transfer; D007073:Immunoglobulin E / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007700:Kinetics; D007942:Leukemia, Experimental / Q000276:immunology; D051381:Rats; D011961:Receptors, Fc / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011961:Receptors, Fc; D007073:Immunoglobulin E