Reversal of the biological activity of Escherichia coli heat-stable enterotoxin by disulfide-reducing agents

Abstract

Various disulfide-reducing agents, mostly thiols and thiol precursors, were examined for their ability to reduce the disulfide bonds in the Escherichia coli heat-stable enterotoxin STa; reduction of the bonds results in loss of biological activity. The biological activity measured was the stimulation of guanylate cyclase in pig intestinal brush border membranes by STa. Nearly all of the compounds inactivated STa, although at different rates; a smaller number appreciably decreased guanylate cyclase activity when they were introduced into the reaction mixture after STa bound to its receptor. With dithiothreitol, the decrease in reaction rate was both time and concentration dependent and resulted in a reversal to basal activity. The anionic thiols were relatively ineffective in reversing activation, the neutral monothiols were moderately effective, and the aminothiols and neutral dithiols were the most effective. The order of effectiveness of the compounds was S-2-(3aminopropylamino)ethanethiol > 2,3-dimercaptopropanol = 2-aminoethylisothiuronium bromide > dithiothreitol > 2-mercaptoethylamine > ax-thioglycerol. These compounds were used in weanling pig ligatedintestinal-loop bioassays to determine if STa-induced secretion was reduced when they were injected 20 min after the STa. Instead of S-2-(3-aminopropylamino)ethanethiol we used the phosphorylated derivative S-2-(3-aminopropylamino)ethylphosphorothioic acid; this compound and 2,3-dimercaptopropanol were the only compounds that reduced STa-induced secretion and had no direct secretory or pathological effects.The heat-stable enterotoxin STa, which is secreted by pathogenic strains of Escherichia coli, induces secretory diarrhea in humans and animals (26). STa is a family of closely related peptides, each containing 18 or 19 amino acids (5, 7,31,42,48), and is probably synthesized as a 72-residue precursor (35,47). All of the deduced structures have six half-cystine residues in identical postions; a 14amino-acid analog containing the sequence has been synthesized and was even more potent than the natural toxin (6). Reduction of the disulfide linkages destroys biological activity (16,42,48).There is an STa receptor located in the brush border of the intestinal epithelium (20, 23); the brush border is also the location of the majority of mature-enterocyte guanylate cyclase (15,55). Field et al. (18), and later Rao et al. (38) and Guerrant et al. (29), showed that STa activated this brush border membrane guanylate cyclase but not other soluble or particulate guanylate cyclases (29, 38). DeJonge (14) and Rao and Field (37) recently reviewed the evidence supporting the hypothesis that this activation is the initial event leading to intestinal ion secretion. Based on this hypothesis, Greenberg et al. examined the effects of disulfide and thiol compounds on STa-induced secretion (25). The disulfide compounds cystamine and cystine, which are capable of reversibly inactivating purified lung guanylate cyclase by mixed disulfide formation (11), decreased STa activation of r...

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

“…This requirement may reflect mixed disulfide formation of ST with low affinity receptors. Indeed, an involvement of critical sulfhydryl groups in toxin-receptor binding has been suggested previously (Waldman et al, 1986;ElDeib et al, 1986).…”

Pharmacological studies on STb heat-stable enterotoxin in pig and rat jejunum

“…This unique structure is responsible for the heat and acid stability of the toxin and its sensitivity to reducing agents (Staples et al, 1980). Disulfide-reducing agents such as thiols have been shown to reduce the disulfide bonds in STa resulting in the loss of biological activity and reversal of the activation of guanylate cyclase (Eldeib et al, 1986).…”