A MONOCLONAL-ANTIBODY DIRECTED AGAINST THE CATALYTIC SITE OF BACILLUS-ANTHRACIS ADENYLYL CYCLASE IDENTIFIES A NOVEL MAMMALIAN BRAIN CATALYTIC SUBUNIT

A brain adenylyl cyclase was shown to contain an epitope closely relat ed to that specified by a conserved sequence containing a nucleotide-b inding consensus sequence GXXXXGKS and located in the catalytic sites of bacterial, calmodulin-dependent adenylyl cyclases [Goyard, S., Orla ndo, C., Sabatier, J.-M., Labruyere, E., d'Alayer, J., Fontan, G., van Rietschoten, J., Mock, M., Danchin, A., Ullmann, A., & Monneron, A. ( 1989) Biochemistry 28, 1964-1967]. A monoclonal antibody, mab 164, pro duced against a peptide corresponding to this conserved sequence speci fically inhibited the Bordetella pertussis adenylyl cyclase. It also s pecifically inhibited rat and rabbit brain synaptosomal adenylyl cycla ses. The extent of inhibition depended upon the type of enzyme purific ation, reaching 90% for the calmodulin-sensitive species of enzyme and 20-35% for the forskolin-agarose-retained species. The extent of inhi bition in a given fraction also depended upon the effector present. ma b 164 reacted on Western blots of forskolin-agarose-retained fractions with a 175-kDa component and did not recognize the Gs-alpha stimulato ry subunit. Consequently, the 175-kDa protein was considered as a good candidate for an adenylyl cyclase catalyst. The adenylyl cyclase acti vity contained in forskolin-agarose-retained fractions was further pur ified on calmodulin-Sepharose. On Western blots of such fractions, mab 164 reacted with a 140-kDa protein, a component that appeared to deri ve from the 175-kDa protein enriched in the previous step. The k(cat) of this 140-kDa presumptive adenylyl cyclase was estimated to be of th e order of 600 s-1. The cloned mammalian adenylyl cyclases of type I [ Krupinski, J., Coussen, F., Bakalyar, H. A., Tang, W.-J., Feinstein, P . G., Orth, K., Slaughter, C., Reed, R. R., & Gilman, A. G. (1989) Sci ence 244, 1558-1564] and type III [Bakalyar, H. A., & Reed, R. R. (199 0) Science 250, 1403-1406] constitute a family which does not contain the GXXXXGKS sequence. The k(cat) of the type I enzyme being lower tha n the one estimated for the catalyst described here; the possibility t hat the latter belongs to another family of adenylyl cyclases is discu ssed.