A brain synaptosomal adenylyl cyclase of high specific activity is photolabeled with azido-ATP.

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.