Invasive adenylyl cyclase of Bordetella pertussis. Physical, catalytic, and toxic properties.
A rapid two-step purification to homogeneity of the calmodulin-activated adenylyl cyclase from urea extracts of Bordetella 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 mumol/mg of enzyme protein/mg of 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 sodium dodecyl sulfate-polyacrylamide gel electrophoresis can be ascribed to anomalous migration. Half-maximal cyclase activation occurs at 3-4 X 10(-10) M calmodulin in the presence of Ca2+ and at 2 X 10(-8) M calmodulin in its absence. Ca2+ activation is maximal at 60-100 microM free CaCl2 (at low calmodulin concentrations), and free Ca2+ concentrations above approximately 125 microM are inhibitory at any calmodulin concentration. Extracellular Ca2+ is essential for intoxication. In Chinese hamster ovary cells, exogenous calmodulin does not inhibit penetration of the cyclase.
Gentile
F
F
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Knipling
L G
LG
Sackett
D L
DL
Wolff
J
J
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001886:Bordetella pertussis / Q000201:enzymology* / Q000472:pathogenicity; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology*; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D014508:Urea; D014774:Virulence
D002147:Calmodulin; D014508:Urea; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine