Localisation of an ATP-binding site on adenylyl cyclase type I after chemical and enzymatic fragmentation

Photolabeling of partially purified bovine brain adenylyl cyclase (AC I) with [gamma(32)P]8-N-3-ATP led to incorporation of P-32 into the 115 kDa catalyst, Further treatment with N-chlorosuccinimide, which cleaves proteins at tryptophan residues, yielded a 14 kDa P-32-labeled fragment. The latter was immunoprecipitated by antibody BBC1, recognizing the extreme C-terminus of AC I, but not by antibody BBC2, recognizing a more remote epitope, Further fragmentation of photolabeled AC I by the proteases Glu-C and Asp-N yielded P-32-labeled peptides corresponding to 2.9 kDa and 5.6 kDa fragments, which were not recognized by any of these antibodies, This narrows the ATP binding site down to a 25 amino acid sequence containing a general motif G(X(0-7))KG(X(0-4))L/M(X(5-7))S/T present in all eukaryotic adenylyl cyclases so far cloned, but also in a variety of bacterial adenylyl cyclases (Peterkofsky et al. (1993) Progr. Nucleic Acids Res. Mol. Biol. 44, 31-65).