ABSTRACT
A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface. The Ricinus communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1. Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin. Displacement of RCA120 from brush border surfaces was proportional to the concentration of B subunit added. This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface. The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine. SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay. The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface. We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside GM1 ligand.
Subject(s)
Cholera Toxin/chemistry , Ileum/metabolism , Microvilli/metabolism , Plant Lectins , Animals , Binding, Competitive , Cholera Toxin/metabolism , G(M1) Ganglioside/metabolism , Hydrogen-Ion Concentration , In Vitro Techniques , Jejunum/metabolism , Lectins/metabolism , Liposomes , Macromolecular Substances , Structure-Activity Relationship , Succinimides/chemistry , Sulfhydryl Compounds/chemistry , SwineABSTRACT
The B subunit of cholera toxin has been covalently attached to the surface of liposomes made from a mixture of phosphatidylethanolamine, phosphatidylcholine and cholesterol. Adenylate cyclase inhibitors and chloride conductance inhibitors were encapsulated within the liposomes. These "targeted" liposomes were used to study the combined effects of this novel delivery system, and a limited number of possible antisecretory agents, on net fluid flux into the pig jejunum. A state of net secretory fluid flux was induced in isolated jejunal loops in weanling pigs by adding theophylline or cholera toxin to the lumen of the isolated loops. There was no reduction in net fluid secretion when liposome suspensions without encapsulated secretory inhibitors were added to fluid in the lumen of loops treated with theophylline. There was also no reduction in net fluid secretion when miconazole, alpha-phenylcinnamate or 5 nitro-2-(3-phenethylamino)benzoate were encapsulated within targeted liposomes added to isolated jejunal loops. The net fluid flux induced by exposure of jejunal loops to theophylline was significantly reduced by adding targeted liposomes containing 2'-deoxy-3'-AMP. The reduction involved a reversal of net secretory fluid flux to an absorptive value. The net fluid secretory response to treatment of loops with cholera toxin was also inhibited by treating loops with targeted liposomes containing 2'-deoxy-3'-AMP. However, the reversal of secretion was less complete for secretion induced by cholera toxin than for secretion induced by theophylline. The reduced antisecretory efficacy versus cholera toxin was not improved by encapsulating higher concentrations of 2'-deoxy-3'-AMP.(ABSTRACT TRUNCATED AT 250 WORDS)
