ADENYLATE-CYCLASE TOXIN FROM BORDETELLA-PERTUSSIS - IDENTIFICATION AND PURIFICATION OF THE HOLOTOXIN MOLECULE

Abstract

Bordetella pertussis adenylate cyclase (AC) toxin is a calmodulin-activated adenylate cyclase enzyme which has the capacity to enter eukaryotic target cells and catalyze the conversion of endogenous ATP into cyclic AMP. In this work, the AC holotoxin molecule is identified and isolated. It is a single polypeptide of apparent 216 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies which immunoprecipitate AC activity from extracts of wild type B. pertussis (BP338) react with this 216-kDa band on Western blots, and it is absent from a transposon Tn5 mutant (BP348) specifically lacking AC toxin. Isolation of the 216-kDa protein to > 85% purity by hydrophobic chromatography, preparative sucrose gradient dnetrifugation, and affinity chromatography using either calmodulin-Sepharose or monoclonal antibody coupled to Sepharose 4B yields stepwise increases in AC toxin potency, to a maximum of 88.3 .mu.mol of cAMP/mg of target cell protein/mg of toxin. Electroelution of the 216-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis yields a preparation with both AC enzyme and toxin activities. These data indicate that this protein represents the AC holotoxin molecule.