Stimulation of adenylate cyclase in washed pigeon erythrocyte membrane with cholera toxin and its subunits.

Cholera toxin was found to stimulate adenylate cyclase activity in washed membrane of pigeon erythrocytes in the presence of dithiothreitol and NAD. When tested with isolated cholera toxin components, the stimulatory activity was found with subunit A or polypeptide A1 derived from this subunit, but not with A2 or subunit B. On a molar basis, polypeptide A1 was approximately four times more active than cholera toxin. Dithiothreitol was not required in the action of polypeptide A1, suggesting that the reagent was needed only to release A1 from subunit A or the holotoxin for their action on adenylate cyclase. The single SH group in polypeptide A1 was not involved in the activity of the peptide, since chemical modification of the thiol group did not alter the stimulatory activity of the peptide. The presence of NAD was, however, essential for the activation of adenylate cyclase with cholera toxin, subunit A, or polypeptide A1. Elevation of the adenylate cyclase activity was also observed when the intact pigeon erythrocytes were incubated with polypeptide A1, although a 30-fold molar excess of A1 over that of holotoxin was required for the same level of activation.