title: Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis.
abstract: An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca2+ to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca2+ and this interaction may be important for its invasion into animal cells. Finally, the technique for purifying the catalytic subunit by SDS-polyacrylamide gel electrophoresis may prove useful in studying the interaction of the adenylyl cyclase with other components produced by the bacteria, as well as the interaction of the enzyme with eukaryotic target cells.
authors.forename: H R
authors.initials: HR
authors.lastname: Masure
authors.forename: M G
authors.initials: MG
authors.lastname: Donovan
authors.forename: D R
authors.initials: DR
authors.lastname: Storm
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D001886:Bordetella pertussis / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange / Q000379:methods; D000242:Cyclic AMP / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D011865:Radioisotope Dilution Technique; D014316:Tritium; D014774:Virulence
chemical_list: D007202:Indicators and Reagents; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D014316:Tritium; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases