Identification and characterization of a new family of high-affinity receptors for Escherichia coli heat-stable enterotoxin in rat intestinal membranes

• Published in: Biochemistry (Easton) Vol. 30; no. 44; pp. 10738 - 10745
• Main Authors: Hugues, Michel, Crane, Marlys, Hakki, Shereen, O'Hanley, Peter, Waldman, Scott A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.11.1991
• Subjects: Animals; Bacterial Toxins - metabolism; Binding Sites; Binding, Competitive; Biological and medical sciences; Cell Membrane - chemistry; Cell Membrane - metabolism; Cell receptors; Cell structures and functions; enterotoxin; Enterotoxins - metabolism; Escherichia coli; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Guanylate Cyclase - metabolism; Intestinal Mucosa - metabolism; Intestines - chemistry; Kinetics; Miscellaneous; Molecular and cellular biology; Rats; Receptors, Cell Surface - analysis; Receptors, Cell Surface - metabolism; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Sodium Chloride - pharmacology; Vertebrata; Membrane receptor; Mammalia; Binding capacity; Rat; Rodentia; Gut; Enterotoxin; Dissociation constant; Scatchard plot; Binding site
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00108a019

Novel high-affinity, low-capacity binding sites in intestinal membranes for the heat-stable toxin produced by Escherichia coli have been defined. The appearance of these sites is observed in the presence of physiological concentrations of NaCl in binding reactions. Scatchard analyses of equilibrium binding in the absence of NaCl demonstrated a single class of binding sites with KD = 1.9 x 10(-9) M and Bmax = 0.75 pmol/mg of protein. In contrast, similar experiments in the presence of NaCl demonstrated, in addition to the previously described low-affinity site, a high-affinity site with a KD of 2.1 x 10(-11) M and a Bmax of 73 fmol/mg of protein. Confirmation of the presence of high- and low-affinity sites was obtained in studies of the kinetics of ST binding. These sites exhibited similar dissociation but markedly different association kinetics. Determination of the association and dissociation constants permitted calculation of the KD's for the high- and low-affinity sites, which were 1.15 x 10(-11) M and 1.89 x 10(-9) M, respectively. These data agree closely with those obtained in studies of equilibrium binding. Furthermore, similar values for the KD's of these sites were obtained in experiments of competitive displacement of labeled ST, confirming the presence of two receptors for this toxin. Binding of ST to high-affinity sites is completely reversible and does not appear to be coupled to activation of particulate guanylate cyclase. In contrast, binding of ST to low-affinity sites appears to be partially reversible and may be coupled to activation of guanylate cyclase.