Activation of the calcium/calmodulin-dependent guanylate cyclase from Paramecium by polypeptide antibiotics and melittin

The activity of the calcium/calmodulin-regulated guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from Paramecium was stimulated by several polypeptides. The most potent activator was melittin (6-fold at 30 μM), followed by alamethicin, suzukacillin, trichotoxin and gramicidin S. Marginal effects were seen with herbicolin A and polymyxin B, whereas the following compounds had no effect: ionophore A23187, actinomycin C₁, destomycin A, gramicidin A, iturin A, nigericin, nonactin, Tü 1718B, valinomycin and synthetic peptide analogues of alamethicin. Guanylate cyclase activation was not related to ion-transport capacity or to the length of the α-helical segments. Rather, the degree of amphiphilicity seemed to be an important criterion. No difference in activation was seen between native guanylate cyclase and the reconstituted enzyme. Thus, in all likelihood, polypeptide stimulation requires the presence of the guanylate cyclase/calmodulin holo-enzyme. Guanylate cyclase activation was permanent. Enzyme kinetics, such as Michaelis-Menten behavior and non-cooperativity, were retained. Incubation with polypeptides at 37°C prior to substrate addition decreased enzyme stimulation. Activation of cGMP formation as enhanced at elevated incubation temperatures. The activation energy for hemolysis of erythrocytes favorably correlated with the extent of guanylate cyclase activation (r = 0.98), suggesting a similar mechanism of interaction with membrane constituents for both processes.