Vol. 130, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS July 31, 1985 Pages 835-840 COMPARISON OF WATER EXPOSED AREA OF CHOLERA TOXIN WHEN FREE IN SOLUTION AND BOUND TO LIPOSOMES CONTAINING THE GANGLIOSIDE GM1 Maurizio TOMASI*, Khalid M. ABU-SALAH**, and John B.C. FINDLAY*** *Laboratorio di Biologia Cellulare, Istituto Superiore di Sanitl, Viale Regina Elena 299, 00161 Roma, Italy **King Saud University, P.O. Box 2455, Biochemistry Department, Riyadh, Saudi Arabia ***Biochemistry Department, The University of Leeds, Leeds LS2 9JT, U.K. Received May 6, 1985 Membrane impermeable diazocoupling reagents were used for studying the water exposure of subunits ( a, B ,y ) of cholera toxin (CT) when bound to liposomes containing the ganglioside G interaction between CT with Lip-GM1 shielded the Mt!ind?r$%l,',bn ':E particular, since a maximum of one amino acid residue on each/l subunit was modifiable. When CT was labeled free in solution five residues of each /? subunit can be coupled, but it produced loss of binding ability. New area ofp subunit was exposed to reagents after having removed a subunit. This labeling may serve as a tool to assess the topology of CT upon binding with Lip-GMl. 0 1985 Academic Press, Inc. Cholera toxin (CT) is a multisubunit protein composed of three different types of subunit: a(MW 21000), p (MW 11400) and y (MW 9000) assembled as a aYp5 oligomer in which the a subunit is linked to the y subunit via a disulfide bond which can be splitted upon treatment with thiol reagents (1). CT initiates cell intossication by a rapid binding between p, region and the cell surface monosialoganglioside GM1 (2, 3). After a lag time of about 20' a subunit modifies irreversibly the adenylate cyclase system which is located in the cytoplasmic side of the plasmamembrane (l-3). The way whereby a subunit crosses the membrane barrier is still unclear. To understand how the interaction between CT and G Ml may induce translocation of a subunit membrane model systems have been used (4-6). In this report we examine the availability of the CT subunits to react with water soluble probes before and after binding with liposomes containing GM1 (Lip-GM1). The reagents employed 0006-291X/85 $1.50 835 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.