The adenylyl cyclase from dormant spores of Phycomyces blakesleeanus is a Type I-like enzyme

Adenylyl cyclase activity was detected in a mixed-membrane fraction from do rmant spores of Phycomyces blakesleeanus. This enzymatic activity increased linearly as a function of protein concentration up to 300 mu g of protein 100 mu l(-1) and 20 min incubation at 25 degrees C. It used Mn2+ or Mg2+ in discriminately as a cofactor, and the addition of both cations together did not have a synergistic effect. The crude enzyme showed a Km,,, for ATP of 0.25 mM, when measured in the presence of Mg2+. It was stable for 48 h at - 20 degrees, losing 25% of its activity after 72 h. The addition of 10 mu M GTP to the enzymatic assay stimulated the adenylyl cyclase, whereas higher concentrations (500 mu M) inhibited it. Cholera toxin and 25 mu M forskolin caused a two-fold stimulation of the enzymatic activity. The calcium-calmo dulin complex stimulated activity two-fold; this stimulation was inhibited by the anti-calmodulin drug trifluoperazine. The enzyme could not be solubi lized by NaCl, but was partially solubilized with non-ionic detergents, ind icating that the enzyme is an integral membrane protein. The detergent-solu bilized enzyme only used Mg2+ as a divalent cation and was also stimulated by calcium-calmodulin, low concentrations of GTP, cholera toxin and forskol in, but was extremely unstable. These results suggest that the adenylyl cyc lase present in dormant spores of Phycomyces blakesleeanus is an integral T ype I-like membrane enzyme.