Characterization of the bacterial cell associated calmodulin-sensitive adenylate cyclase from Bordetella pertussis.
Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase that is associated with the whole bacteria and released into its culture media. Preparations of this enzyme invade animal cells, causing elevations in intracellular cAMP levels. Cell-associated adenylate cyclase accounted for 28% of the total adenylate cyclase activity while 72% was released into the culture supernatant. Over 90% of the cell-associated adenylate cyclase activity was sensitive to trypsin treatment of whole cells, indicating that the catalytic domain of the enzyme is localized on the outer surface of the bacterial cells. Enzyme activity was released from whole cells by treatment with SDS. This activity was resolved as a large form (Mr 215,000) by SDS-polyacrylamide gel electrophoresis. In contrast, the culture supernatant contained only the 45,000-dalton catalytic subunit. Enzyme activity released from spheroplasts by sonication was resolved into a large form (Mr 215,000) and a small form (Mr 45,000). The appearance of the small form with spheroplast formation was probably the result of proteolytic degradation. Antibodies generated against the catalytic subunit purified from culture supernatants cross-reacted with and immunoprecipitated both the large and small forms of adenylate cyclase isolated from bacterial cells. Furthermore, incubation of the cell-associated enzyme with a crude bacterial extract resulted in a time-dependent disappearance of the 215,000-dalton form and a concomitant increase in the amount of the smaller 45,000-dalton form. There was also a parallel increase in the ability of the cell-associated preparation to elevate intracellular cAMP levels in N1E-115 mouse neuroblastoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Masure
H R
HR
Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.
Storm
D R
DR
D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001886:Bordetella pertussis / Q000201:enzymology*; D002147:Calmodulin / Q000494:pharmacology*; D002460:Cell Line; D000242:Cyclic AMP / Q000032:analysis; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009447:Neuroblastoma / Q000032:analysis; D013104:Spheroplasts / Q000201:enzymology
D002147:Calmodulin; D007527:Isoenzymes; D000242:Cyclic AMP; D000262:Adenylyl Cyclases