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.
Castets
F
F
Départment des Protéines de la Transduction, LCB, CNRS, Marseille, France.
Baillat
G
G
Mirzoeva
S
S
Mabrouk
K
K
Garin
J
J
d'Alayer
J
J
Monneron
A
A
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001386:Azides / Q000378:metabolism*; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002147:Calmodulin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D051381:Rats; D013574:Synaptosomes / Q000201:enzymology*
D000345:Affinity Labels; D000906:Antibodies; D001386:Azides; D002147:Calmodulin; D010455:Peptides; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D004533:Egtazic Acid; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases