Activation of intestinal brush border guanylate cyclase by aromatic disulphide compounds

• Published in: Biochemical journal Vol. 275; no. 1; pp. 29 - 34
• Main Authors: ELDEIB, M. M. R, PARKER, C. D, WHITE, A. A
• Format: Journal Article
• Language: English
• Published: Colchester Portland Press 01.04.1991
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Bacterial Toxins - pharmacology; Biological and medical sciences; brush border membranes; Disulfides - metabolism; Disulfides - pharmacology; Dithionitrobenzoic Acid - pharmacology; Enterotoxins - pharmacology; Enzyme Activation - drug effects; Enzymes and enzyme inhibitors; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; guanylate cyclase; Guanylate Cyclase - metabolism; Intestines - enzymology; Intestines - ultrastructure; Lyases; Male; Microvilli - drug effects; Microvilli - enzymology; Nicotinic Acids - pharmacology; Polyethylene Glycols - pharmacology; Sulfhydryl Compounds - metabolism; Swine; Thioguanine - analogs & derivatives; Thioguanine - pharmacology; thiouracil; Rat; Enzyme; Thiohydroxyl group; Rodentia; Gut; Activation; Molecular exchange; Guanylate cyclase; Dose activity relation; Toxin; Vertebrata; Brush border; Membrane receptor; Mammalia; Enzymatic activity; Disulfide bond; Molecular complex; Aromatic compound; Mechanism of action
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2750029

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.