A rapid two-step purification to homogeneity of the calmodulin-activated adenylate cyclase from urea extracts of Bordella pertussis organisms (strain 114) is described. Catalytic and invasive activities are purified 30- and 177-fold respectively, and virtually no degraded forms are found. Specific activities are 0.4 mmol/min/mg and 0.5 micromol/mg enzyme protein/mg cell protein/min for catalytic and invasive activities, respectively. The 15 amino terminal amino acids agree with those deduced from the DNA sequence, as does the molecular mass of 175 kDa (guanidine) or 177 kDa (urea) obtained by equilibrium sedimentation. The larger apparent molecular mass seen in SDS PAGE can be ascribed to anomalous migration.@ Half maximal cyclase activation occurs at 3-4xlO(-10) M calmodulin in the presence of Ca(2+) and at 2xlO(-8) M calmodulin in its absence. Ca(2+) activation is maximal at 60-100 microM free CaCl(2) (at low calmodulin concentrations), and free Ca(2+) concentrations above 125 microM are inhibitory at any calmodulin concentration. Extracellular Ca(2+) is essential for intoxication. In CHO cells exogenous CaM does not inhibit penetration of the cyclase.
