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.(ABSTRACT TRUNCATED AT 250 WORDS)

Active receptors for Escherichia coli heat-stable enterotoxin (ST) were partially purified by ligand-affinity chromatography. The affinity column was prepared by coupling ST to biotin derivatized with an extended N-hydroxysuccinylated spacer arm prior to binding to monomeric avidin immobilized on agarose. Detergent extracts of rat intestinal mucosa membranes were quantitatively depleted of ST binding activity when chromatographed on this affinity matrix. Biotinylated ST-receptor complexes were eluted from affinity columns with 2 mM biotin and these complexes quantitatively dissociated with bile salts. Using this technique, functional ST receptors were purified maximally about 2000-fold, with about 3% of the total activity in crude extracts recovered in these purified preparations. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis and silver staining demonstrated a major protein subunit of 74 kDa. Affinity cross-linking of these preparations to 125I-ST demonstrated specific labeling predominantly of the 74-kDa subunit. In addition, lower amounts of labeled ST were incorporated into subunits of 164 and 45 kDa, confirming the heterogeneous nature of ST receptors. Purified receptors bound ST in a concentration-dependent fashion, with an IC50 of 10(-9) M. These studies demonstrate that ligand-affinity chromatography can be employed to purify ST receptors. The availability of purified receptors will facilitate further studies of mechanisms underlying ST-induced intestinal secretion.

Summary.A procedure was developed for enumeration of total associated, attached and intracellular bacteria after interaction of Yersinia spp. with epithelial cells in vitro. Isogenic cultures of Y. enterocolitica grown at 25°C had greater affinity for epithelial cells (Henle, HeLa and Vero) than for polystyrene, and they invaded the cells. Y. kristenseni and Y. intermedia showed less attachment to either surface and were non-invasive. The degree of attachment to cells and invasion by Y . enterocolitica was related to number of bacteria added and interaction time, whereas attachment to polystyrene occurred rapidly and did not change. Y. enterocolitica was more hydrophobic when grown at 35°C than at 25°C according to partitioning in a biphasic dextran-polyethylene glycol system, and attached strongly to both polystyrene and epithelial-cell monolayers. Y. kristenseni grown at 25°C was also hydrophobic but did not have the same attachment properties. Y. kristenseni and Y . intermedia showed slightly reduced electrostatic interactions with the anion exchangers DEAE-Sepharose and DEAE-Trisacryl. Attachment of Y. enterocolitica to epithelial cells probably involves non-specific surface properties that are not entirely explicable by hydrophobic and electrostatic interactions, whereas invasion of epithelial cells appears to resemble "receptor-mediated endocytosis".

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