A BRAIN SYNAPTOSOMAL ADENYLYL-CYCLASE OF HIGH SPECIFIC ACTIVITY IS PHOTOLABELED WITH AZIDO-ATP

Partially purified adenylyl cyclase preparations of high specific acti vity (60 +/- 10 mu mol cAMP/ (mg.min)) were obtained from rat brain sy naptosomes (Orlando, C., d'Alayer, J., Baillat, G., Castets, F., Jeann equin, O., Mazie, J. C., and Monneron, A. (1992) Biochemistry 31, 3215 -3222). Adenylyl cyclase activity was stimulated 4-fold by Ca2+/calmod ulin and 2-fold by forskolin or by Mn2+. These preparations contained two major proteins of 140 and 110 kDa. The 140-kDa protein was identif ied as the neural cell adhesion molecule. The 110-kDa protein was spec ifically 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-depen dent manner and was by far the most heavily labeled component of FC fr actions. Saturation was obtained with 30 mu M [P-32]8-N(3)ATP. Photoin sertion of N(3)ATP into the protein was largely prevented by ATP or ad enylyl imidodiphosphate but not by ADP, AMP, or adenosine. A modest in corporation 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 exp ected from adenylyl cyclase type I, it appears that its specific activ ity is on the order of 1 mmol cAMP/(mg.min), about 200-fold that measu red for brain adenylyl cyclases type I.