Nucleotide regulation of heat-stable enterotoxin receptor binding and of guanylate cyclase activation.
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.
Katwa
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LC
John M. Dalton Research Center, University of Missouri-Columbia 65211.
Parker
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Dybing
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White
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AA
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000227:Adenine Nucleotides / Q000494:pharmacology; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism* / Q000494:pharmacology; D004768:Enterotoxins / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008871:Microvilli / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013552:Swine; D013873:Thionucleotides / Q000494:pharmacology
D000227:Adenine Nucleotides; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D006150:Guanine Nucleotides; D009711:Nucleotides; D011956:Receptors, Cell Surface; D013873:Thionucleotides; C034151:heat stable toxin (E coli); D006153:Guanosine Diphosphate; C032829:6-thioguanosine 5'-diphosphate; D000246:Adenosine Diphosphate Ribose; D000266:Adenylyl Imidodiphosphate; D002731:4-Chloromercuribenzenesulfonate; D006162:Guanylate Cyclase; D008274:Magnesium