Open AccessCytosolic activator of adenylate cyclase: Reconstitution, characterization, and mechanism of action.
Author(s) -
Naji Sahyoun,
Harry LeVine,
P J Stenbuck,
Pedro Cuatrecasas
Publication year - 1983
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.80.12.3646
Subject(s) - cyclase , adenylate kinase , cytosol , activator (genetics) , gtp' , biochemistry , biology , enzyme , chemistry , receptor
Rat liver and isolated hepatocytes contain high levels of a soluble adenylate cyclase stimulator, whereas rat erythrocytes lack this activity. Accordingly, a reconstitution system was developed with adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from erythrocyte ghosts and the soluble activator from liver cytosol. Pretreatment of erythrocyte ghosts with the cytosolic factor resulted in a 5- to 15-fold activation of adenylate cyclase in the presence or absence of NaF, 5'-guanylyl imidodiphosphate, or isoproterenol and GTP. The sequence of addition of the cytosolic component and the other activators was critical in determining the maximal activity of the enzyme. The cytosolic factor appears to be a heat-labile Mr 105,000 protein, which activates adenylate cyclase in a saturable reaction involving binding of the protein to the erythrocyte ghosts. This molecular interaction was accompanied by stabilization of a labile thiol group that was essential for catalytic activity. The cytosolic component also unmasks latent adenylate cyclase activity in human erythrocyte ghosts and in cytoskeletal preparations from rat erythrocyte ghosts. These observations suggest that the cytosolic activator may also occur as a native, peripheral membrane component of adenylate cyclase systems and may be required for the expression and stabilization of catalytic activity.