A Brain Synaptosomal Adenylyl Cyclase of High Specific Activity Is Photolabeled with Azido-ATP

• Published in: Biochemistry (Easton) Vol. 33; no. 17; pp. 5063 - 5069
• Main Authors: Castets, Francis, Baillat, Gilbert, Mirzoeva, Salida, Mabrouk, Kamel, Garin, Jerome, d'Alayer, Jacques, Monneron, Ariane
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01-05-1994
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Adenylyl Cyclases - isolation & purification; Adenylyl Cyclases - metabolism; Affinity Labels - metabolism; Amino Acid Sequence; Animals; Antibodies; Azides - metabolism; Blotting, Western; Brain - enzymology; Calmodulin - pharmacology; Colforsin - pharmacology; Egtazic Acid - pharmacology; Electrophoresis, Polyacrylamide Gel; Guanylyl Imidodiphosphate - pharmacology; Kinetics; Manganese - pharmacology; Molecular Sequence Data; Molecular Weight; Peptides - chemical synthesis; Peptides - immunology; Rats; Synaptosomes - enzymology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00183a009

Partially purified adenylyl cyclase preparations of high specific activity (60 +/- 10 mumol cAMP/(mg.min)) were obtained from rat brain synaptosomes (Orlando, C., d'Alayer, J., Baillat, G., Castets, F., Jeannequin, O., Mazié, J. C., & Monneron, A. (1992) Biochemistry 31, 3215-3222). Adenylyl cyclase activity was stimulated 4-fold by Ca2+/calmodulin and 2-fold by forskolin or by Mn2+. These preparations contained two major proteins of 140 and 110 kDa. The 140-kDa protein was identified as the neural cell adhesion molecule. The 110-kDa protein was specifically recognized by affinity-purified antibodies directed against a peptide corresponding to sequence 976-1013 of adenylyl cyclase type I. It was photolabeled by [alpha-32P]8- and 2-N3ATP in a light-dependent manner and was by far the most heavily labeled component of FC fractions. Saturation was obtained with 30 microM [32P]8-N3ATP. Photoinsertion of N3ATP into the protein was largely prevented by ATP or adenylyl imidodiphosphate but not by ADP, AMP, or adenosine. A modest incorporation of N3cAMP and photoinsertion of [alpha-32P]N3GTP into the 110-kDa protein were observed. Although some of the properties of the synaptosomal 110-kDa protein described here would match those expected from adenylyl cyclase type I, it appears that its specific activity is on the order of 1 mmol cAMP/(mg.min), about 200-fold that measured for brain adenylyl cyclases type I.