Activation of intestinal brush border guanylate cyclase by aromatic disulphide compounds.
Guanylate cyclase in pig intestinal brush border membranes was stimulated by certain aromatic disulphides. The most effective were 6-thioguanine disulphide [(TGS)2], 6-mercaptopurine disulphide, 6,6'-dithiodinicotinic acid, 5,5'-dithiobis-(2-nitrobenzoic acid) and 5-carboxy-2-thiouracil disulphide. (TGS)2 stimulated activity 15-fold when present at 0.1 mM. The optimum concentration for each disulphide was different, and higher concentrations were inhibitory. There was no activation by alkyl disulphides or by N-ethylmaleimide. Activation by 50 microM-(TGS)2 was partially reversed by later addition of 0.1 mM-dithiothreitol, whereas activation by the Escherichia coli heat-stable enterotoxin STa was relatively unaffected. Pretreatment of the membranes with (TGS)2 produced a concentration-dependent inhibition of STa-stimulated activity, while stimulating basal activity, until the activities were equal at 50 microM. Activity was [Mg2+]-dependent, the optimal [Mg2+] progressively increasing as the enzyme was stimulated by (TGS)2, STa and Lubrol PX respectively. However, (TGS)2 pretreatment prevented the shift to higher [Mg2+]optima induced by STa or Lubrol alone. Substitution of Mn2+ for Mg2+ in the reaction elevated basal activity and eliminated by activation (TGS)2. (TGS)2 only inhibited Mn2(+)-dependent activity (both basal and stimulated). The affinity of 125I-STa for its receptor was slightly increased by (TGS)2. We propose that (TGS)2 undergoes thiol-disulphide exchange with at least three different protein thiols of decreasing reactivity. The first is associated with Mg2(+)-dependent activation, the second is associated with a tonic inhibition of activity and the third is associated with the catalytic activity, although probably not at the active site.
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John M. Dalton Research Center, University of Missouri-Columbia 65211.
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D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004768:Enterotoxins / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000378:metabolism*; D007422:Intestines / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008871:Microvilli / Q000187:drug effects / Q000201:enzymology*; D009539:Nicotinic Acids / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013552:Swine; D013866:Thioguanine / Q000031:analogs & derivatives* / Q000494:pharmacology
D001427:Bacterial Toxins; D004220:Disulfides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D009539:Nicotinic Acids; D013438:Sulfhydryl Compounds; C034151:heat stable toxin (E coli); C001102:6,6'-dithiodinicotinic acid; D011092:Polyethylene Glycols; C005092:lubrol; C068364:6-thioguainine disulfide; D004228:Dithionitrobenzoic Acid; D006162:Guanylate Cyclase; D013866:Thioguanine