Adenylate cyclase activity of a 68,000-molecular-weight protein isolated from the outer membrane of Bordetella bronchiseptica.
A method was developed which is suitable for the isolation of substantial quantities of outer membrane proteins of Bordetella species in a water-soluble form. The extracted material may then be further fractionated in the absence of detergents by ion-exchange chromatography and preparative flat-bed isoelectrofocusing. These procedures facilitated the isolation of one of the proteins, of molecular weight 68,000, for which the antibody titer correlated with the degree of protection against nasal changes induced in specific-pathogen-free piglets by Bordetella bronchiseptica infection (P. Novotny, M. Kobisch, K. Cownley, A. P. Chubb, and J. A. Montaraz, Infect. Immun. 50:190-198). This protein, which banded between 7.0 and 7.6 pH in preparative isoelectrofocusing, may be further purified with a monoclonal immunosorbent. Immunopurified protein showed adenylate cyclase activity. The enzymatic activity was found to be unstable during processing; i.e., although the crude extract showed up to 150 nmol of cyclic AMP per mg/min, the immunopurified protein showed a maximum of only 200 nmol of cyclic AMP per mg/min. Two strains of B. bronchiseptica, isolated from herds of healthy pigs showing no signs of atrophic rhinitis, did not produce the 68,000-molecular-weight protein and were negative for adenylate cyclase. However, it is not known whether the 68,000-molecular-weight protein is a component of adenylate cyclase or whether it is an unrelated protein associated with this enzyme in some unknown way. Adenylate cyclase activity from culture supernatants of B. bronchiseptica, B. pertussis, and B. parapertussis can be absorbed equally to the same monoclonal immunosorbent.
D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001884:Bordetella / Q000201:enzymology*; D002846:Chromatography, Affinity; D007526:Isoelectric Point; D008970:Molecular Weight; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology
D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D000262:Adenylyl Cyclases