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Analysis of Two-Dimensional Dissociation Constant of Laterally Mobile Cell Adhesion Molecules

De-Min Zhu ^*, Michael L. Dustin , Christopher W. Cairo ^* and David E. Golan ^* 

^* Department of Biological Chemistry and Molecular Pharmacology, and Department of Medicine, Harvard Medical School, Hematology Division, Brigham and Women's Hospital, Boston, Massachusetts; and Skirball Institute of Biomolecular Medicine and Department of Pathology, New York University School of Medicine, New York, New York

Correspondence: Address reprint requests to David E. Golan, MD, PhD, Tel.: 617-432-2256; E-mail: dgolan{at}hms.harvard.edu.

We formulate a general analysis to determine the two-dimensional dissociation constant (2D K_d), and use this method to study the interaction of CD2-expressing T cells with glass-supported planar bilayers containing fluorescently labeled CD58, a CD2 counter-receptor. Both CD2 and CD58 are laterally mobile in their respective membranes. Adhesion is indicated by accumulation of CD2 and CD58 in the cell-bilayer contact area; adhesion molecule density and contact area size attain equilibrium within 40 min. The standard (Scatchard) analysis of solution-phase binding is not applicable to the case of laterally mobile adhesion molecules due to the dynamic nature of the interaction. We derive a new binding equation, B/F = [(N_t x f)/(K_d x S_cell)] – [(B x p)/K_d], where B and F are bound and free CD58 density in the contact area, respectively; N_t is CD2 molecule number per cell; f is CD2 fractional mobility; S_cell is cell surface area; and p is the ratio of contact area at equilibrium to S_cell. We use this analysis to determine that the 2D K_d for CD2-CD58 is 5.4–7.6 molecules/µm². 2D K_d analysis provides a general and quantitative measure of the mechanisms regulating cell-cell adhesion.

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