Isolation of a protein fraction from Bordetella pertussis that facilitates entry of the calmodulin-sensitive adenylate cyclase into animal cells.
Bordetella pertussis, the pathogen responsible for whooping cough, releases a soluble calmodulin-sensitive adenylate cyclase into its culture medium. Several investigators have shown that the partially purified adenylate cyclase is capable of entering animal cells and elevating intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24,6323-6328]. However, the mechanism for entry of the catalytic subunit of the adenylate cyclase into animal cells is unknown. Recently, it was determined that the purified catalytic subunit of the enzyme is unable to enter animal cells [Masure, H. R., Oldenburg, D. J., Donovan, M. G., Shattuck, R. L., & Storm, D. R. (1988) J. Biol. Chem. 263, 6933-6940]. On the basis of these data and other observations, we hypothesized that the culture medium of B. pertussis contains one or more additional polypeptides which facilitate entry of the adenylate cyclase catalytic subunit into animal cells. In this study, we report that a cell-invasive preparation of B. pertussis adenylate cyclase was rendered noninvasive after passage through a wheat germ lectin-agarose column. A fraction was eluted from the wheat germ lectin-agarose column with N-acetyl-D-glucosamine. This fraction, when combined with the noninvasive adenylate cyclase, was able to restore the ability of the adenylate cyclase preparation to enter neuroblastoma cells and increase intracellular cAMP levels. Furthermore, the fraction eluted from the wheat germ lectin-agarose column was found to be trypsin and chymotrypsin sensitive, suggesting that this material was proteinaceous.(ABSTRACT TRUNCATED AT 250 WORDS)
Donovan
M G
MG
Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.
Masure
H R
HR
Storm
D R
DR
D000262:Adenylyl Cyclases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D001886:Bordetella pertussis / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002478:Cells, Cultured; D002847:Chromatography, Agarose; D000242:Cyclic AMP / Q000032:analysis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes; D009447:Neuroblastoma / Q000378:metabolism; D014909:Wheat Germ Agglutinins
D001426:Bacterial Proteins; D002147:Calmodulin; D007457:Iodine Radioisotopes; D014909:Wheat Germ Agglutinins; D000242:Cyclic AMP; D000262:Adenylyl Cyclases