Nucleotide regulation of heat-stable enterotoxin receptor binding and of guanylate cyclase activation.

Abstract

Certain nucleotides were found to regulate the binding of the Escherichia coli heat-stable enterotoxin (STa) to its receptor in pig intestinal brush border membranes. ATP and adenine nucleotide analogues inhibited 125I-STa binding, while guanine nucleotide analogues stimulated binding, with maximal effects at 0.5-1.0 mM. The strongest inhibitors were adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) (36%) and adenosine 5'-[beta-thio]diphosphate (ADP[S]) (41%). Inhibition did not require Mg2+, and was blocked by p-chloromercuribenzenesulphonate (PCMBS). Stimulation of binding required Mg2+, was not prevented by PCMBS and was maximal with GDP[S] (41%). While App[NH]p and MgGDP[S] appeared to be acting at different sites, they also interfered with each other. These nucleotides exerted only inhibitory effects on STa-stimulated guanylate cyclase activity, in contrast with the stimulatory effects of adenine nucleotides on atrial natriuretic peptide (ANP)-stimulated guanylate cyclase. Inhibition by low concentrations of MgApp[NH]p and MgATP was weaker above 0.1 mM, while MgGDP[S] and magnesium guanosine 5'-[gamma-thio]triphosphate (MgGTP[S]) inhibited in a single phase. Inhibition by MgApp[NH]p, at all concentrations, was competitive with the substrate (MgGTP), as was that by MgGDP[S] and MgGTP[S]. Whereas membrane guanylate cyclases usually show positively co-operative kinetics with respect to the substrate, STa-stimulated activity exhibited Michaelis-Menten kinetics with respect to MgGTP. This changed to positive co-operativity when Lubrol PX was the activator, or when the substrate was MnGTP. These results suggest the presence of both a regulatory and a catalytic nucleotide-binding site, which do not interact co-operatively with STa activation.