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 related to that specified by a conserved sequence containing a nucleotide-binding consensus sequence GXXXXGKS and located in the catalytic sites of bacterial, calmodulin-dependent adenylyl cyclases [Goyard, S., Orlando, 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, produced against a peptide corresponding to this conserved sequence specifically inhibited the Bordetella pertussis adenylyl cyclase. It also specifically inhibited rat and rabbit brain synaptosomal adenylyl cyclases. The extent of inhibition depended upon the type of enzyme purification, reaching 90% for the calmodulin-sensitive species of enzyme and 20-35% for the forskolin-agarose-retained species. The extent of inhibition in a given fraction also depended upon the effector present. mab 164 reacted on Western blots of forskolin-agarose-retained fractions with a 175-kDa component and did not recognize the Gs alpha stimulatory subunit. Consequently, the 175-kDa protein was considered as a good candidate for an adenylyl cyclase catalyst. The adenylyl cyclase activity contained in forskolin-agarose-retained fractions was further purified on calmodulin-Sepharose. On Western blots of such fractions, mab 164 reacted with a 140-kDa protein, a component that appeared to derive from the 175-kDa protein enriched in the previous step. The kcat of this 140-kDa presumptive adenylyl cyclase was estimated to be of the order of 600 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Orlando
C
C
Laboratoire de Biochimie, CNRS URA 1455, Faculté de Médecine Secteur Nord, Marseille, France.
d'Alayer
J
J
Baillat
G
G
Castets
F
F
Jeannequin
O
O
Mazié
J C
JC
Monneron
A
A
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D001408:Bacillus anthracis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000276:immunology; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D002417:Cattle; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D051381:Rats; D013574:Synaptosomes / Q000187:drug effects / Q000201:enzymology
D000067956:Adenylyl Cyclase Inhibitors; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000262:Adenylyl Cyclases