Soluble adenylate cyclase from the culture medium of Bordetella pertussis: purification and characterization

Abstract

Culture medium of exponentially growing B. pertussis (strain 114) contains significant quantities of soluble (100,000xg for 1 h) adenylate cyclase. The enzyme was purified by chromatography on diethylaminoethyl cellulose and Sephadex G 200. The purest material yielded a single band on sodium dodecyl sulfate disc gel electrophoresis. It is labile, has a temperature optimum of 30°C, a pH optimum of pH 7 to 8, and a K(m) for adenosine 5' triphosphate of 0.4 mM, and requires Mg2+ for maximum activity. The molecular weight, by sodium dodecyl sulfate disc gel electrophoresis and sucrose density gradient, is approximately 70,000. The enzyme is markedly inhibited by fluoride and weakly inhibited by monovalent salts, but its activity is not altered by α keto acids or nonsubstrate nucleoside triphosphates. Thus, by its presence in the culture supernatant, its smaller molecular weight, and its insensitivity to α keto acids and nucleotides, this enzyme differs from the bacterial adenylate cyclases previously described.