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Localisation of an ATP‐binding site on adenylyl cyclase type I after chemical and enzymatic fragmentation
Author(s) -
Droste Martin,
Mollner Stefan,
Pfeuffer Thomas
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00735-1
Subject(s) - adenylyl cyclase , adcy10 , biochemistry , adcy9 , enzyme , chemistry , microbiology and biotechnology , peptide sequence , epitope , binding site , sequence motif , amino acid , biology , antibody , dna , gene , immunology
Photolabeling of partially purified bovine brain adenylyl cyclase (AC I) with [ γ 32 P8‐ N ‐ATP led to incorporation of 32 P into the 115 kDa catalyst. Further treatment with N ‐chlorosuccinimide, which cleaves proteins at tryptophan residues, yielded a 14 kDA 32 P‐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 32 P‐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.