Mechanism of activation of adenylate cyclase by cholera toxin

Cholera toxin (choleragen) can stimulate adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] activity in whole particulate fractions or purified plasma membranes of homogenates of isolated fat cells provided special precautions are taken to stabilize the enzyme during the required preincubation period. As observed with intact cells, the activation exhibits a protracted (about 25 min) lag phase, and it is blocked by ganglioside GM1 and choleragenoid ("binding" subunit of toxin). The 36,000 molecular weight subunit ("active" subunit), a hydrophobic polypeptide which does not block choleragen binding or action, can directly activate the enzyme in intact cells without a lag phase. Its effects are not blocked by ganglioside GM1 or choleragenoid, yet the stimulated activity exhibits reduced fluoride and enhanced isoproterenol sensitivity, properties characteristic of the choleragen-activated enzyme. Binding of the 125I-labeled 36,000 molecular weight subunit to cells is not saturable and is unaffected by gangliosides, choleragen, or choleragenoid, and the bound material behaves as an integral membrane protein; this protein may simply partition into the membrane matrix. With increasing time of incubation cell-bound choleragen may dissociate into its component subunits, but these remain in the membrane. Using a double antibody immunoprecipitin system, substantial precipitation of cyclase activity occurs with antisera against the 36,000 molecular weight subunit provided toxin activation has occurred. The normal process of activation may involve an initially inactive toxin--ganglioside complex which, as a result of lateral mobility and multivalent binding (lag phase), results in destabilization of the molecule with release of the "active" subunit into the membrane core where it can spontaneously associate with and perturb the cyclase complex.