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 mu mol cAMP/ (mg.min)) were obtained from rat brain synaptosomes (Orlando, C., d'Alayer, J., Baillat, G., Castets, F., Jeannequin, O., Mazie, J. C., and 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-P-32]8- and 2-N(3)ATP in a light-dependent manner and was by far the most heavily labeled component of FC fractions. Saturation was obtained with 30 mu M [P-32]8-N(3)ATP. Photoinsertion of N(3)ATP into the protein was largely prevented by ATP or adenylyl imidodiphosphate but not by ADP, AMP, or adenosine. A modest incorporation of N(3)cAMP and photoinsertion of [alpha-P-32]N(3)GTP 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.