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

Abstract

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.

Localisation of an ATP‐binding site on adenylyl cyclase type I after chemical and enzymatic fragmentation

“…The purified type I adenylyl cyclase used in this study had similar affinities for ATP and 8-N3-ATP which was in contrast to the fndings of Casters et al [6] who found a comparable affinity for 8-Na-ATP (22 gM) but a 5-fold higher affinity for the substrate ATP. However, this difference may be due to a different isoform of adenylyl cyclase for which the authors claimed a > 100-fold higher specific activity than that reported here.…”

“…Unless specified otherwise, adenylyl cyclase in buffer A was concentrated 5-10-fold by centrifugation through a Microcon 30 filter device (Amicon). Solutions were adjusted to 5 mM MnC12 following the protocol of Castets et al [6]. Photolabeling was performed on ice in microtiter plates (30-60 ~tl per well).…”

“…This ATP analog has been previously used by Castets et al [6] for labeling of a partially purified adenylyl cyclase from rat brain synaptosomes. Since photoaffinity labeling usually proceeds with low efficiency and 32p-labeled peptides, due to addition of several negative charges, may migrate differently from unlabeled peptides, precipitation by specific antibodies against adenylyl cyclase was used to check coincidence of labeling and immunoreactivity.…”

A novel calmodulin-binding protein, belonging to the WD-repeat family, is localized in dendrites of a subset of CNS neurons.

“…Mr present in a fraction highly enriched in adenylyl cyclase activity was microsequenced (Castets, F., G. Baillat, S. Mirzoeva, K. Mabrouk, J. Garin, J. d'Alayer, and A. Monneron. 1994.…”

Cardiac striatin interacts with caveolin-3 and calmodulin in a calcium sensitive manner and regulates cardiomyocyte spontaneous contraction rate

“…Striatin (STRN) is a multivalent protein that was originally purified from highly active adenylyl cyclase (AC) rich fractions from rat brain synaptosomes, and was found to bind calmodulin (CaM) in a calcium sensitive manner (Bartoli et al 1998;Castets et al 1994;Castets et al 2000). Both CaM and AC are reportedly regulators of the E-C coupling in the heart and modulators of cardiac contractility (Maier and Bers 2002;Tang et al 2010).…”