The activation of adenylate cyclase by cholera toxin: possible interaction with the nucleotide regulatory site

The application of cholera toxin to intact cells causes a stimulation of adenylate cyclase activity. The effect is characterized by a lag period followed by a progressive rise in enzyme activity over several hours. Only a few minutes' exposure to the toxin is required to produce effects lasting over several days. Stimulation of adenylate cyclase by cholera toxin in broken cell preparations requires the presence of nicotinamide-adenine dinucleotide (NAD) and an unidentified component of the cytosol. Guanyl nucleotides and certain non-hydrolysable analogues of guanosine triphosphate also stimulate adenylate cyclase. Stimulation by the analogues results in a highly activated enzyme which has characterisitcs similar to those of adenylate cyclase after stimulation by cholera toxin. Thus the stimulation is irreversible, the enzyme may be "solubilized" by non-ionic detergents in the activated state, and responses to certain hormones are enhanced. Therefore the possibility exists that cholera toxin acts on the guanyl nucleotide regulatory protein of the adenylate cyclase complex. In exploring this possibility it was found pretreatment with cholera toxin not only blocked the stimulatory effect of subsequently added guanylylimidodi-phosphate (GppNHp) but that the latter reduced the stimulation by toxin. Similarly, pretreatment with GppNHp blocked the effect of cholera toxin. The similarities in the effects of cholera toxin and GppNHp, together with the mutual interference of their activities, suggests that cholera toxin acts at the same regulatory site at which guanyl nucleotides exert their effects on adenylate cyclase.