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Distribution and Mobility of Murine Histocompatibility H-2Kk Antigen in the Cytoplasmic Membrane

Damjanovich, Sándor and Trón, Lajos and Szöllösi, János and Zidovetzki, Raphael and Vaz, Winchil L. C. and Regateiro, Fernando and Arndt-Jovin, Donna J. and Jovin, Thomas M. (1983) Distribution and Mobility of Murine Histocompatibility H-2Kk Antigen in the Cytoplasmic Membrane. Proceedings of the National Academy of Sciences of the United States of America, 80 (19). pp. 5985-5989. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:DAMpnas83

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Abstract

The topographical distributions and mobilities of the murine histocompatibility antigen H-2Kk and of concanavalin A (Con A) binding sites have been studied on a murine lymphoma cell line. The spatial distribution of H-2Kk antigens, the average distance between H-2Kk antigens and Con A binding sites, and the separation of different determinants on the H-2Kk 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-2Kk antibodies conjugated with fluorescein (donor) and rhodamine (acceptor), we conclude that the H-2Kk antigen exists without appreciable clustering on the cell surface. Substantial energy transfer between appropriately labeled Con A and antibodies bound to the H-2Kk antigen shows that the two populations are interspersed. Donor/acceptor pairs of monoclonal antibodies binding to different determinants on the same H-2Kk 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-2Kk 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 µ s 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 cm^2· s^-1 for the H-2Kk antigen labeled with fluoresceinated IgG or its corresponding Fab fragment.


Item Type:Article
Additional Information:Copyright © 1983 by the National Academy of Sciences Communicated by J. Szentágothai, May 23, 1983 Dr. R.M. Clegg, Dr. E.D. Matayoshi, Mr. G. Striker, and Dr. A.F. Corin are acknowledged for their helpful discussions. We thank Mr. S.H. Helliwell for skillful technical assistance in flow cytometric experiments, Ms. Lalande and Ms. von Bogen for assisting with tissue culture. We are indebted to Dr. K. Rajewsky and Dr. B. Holtkamp for helpful discussions of monoclonal antibodies and cell lines. The work was carried out as a part of a research program between the Abteilung Molekulare Biologie; Max-Planck-Institut fur Biophysikalische Chemie, Gottingen, Federal Republic of Germany; and the Department of Biophysics, Medical-University School of Debrecen, Debrecen, Hungary, jointly sponsored by the Deutsche Forschungsgemeinschaft and the Hungarian Academy of Sciences. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisenent" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Subject Keywords:rotational diffusion; translational diffusion; energy transfer; phosphorescence; flow cytometry
Record Number:CaltechAUTHORS:DAMpnas83
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:DAMpnas83
Alternative URL:http://www.pnas.org/cgi/content/abstract/80/19/5985
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6985
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:04 Jan 2007
Last Modified:26 Dec 2012 09:27

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