Effects of nucleoside triphosphates on choleragen-activated brain adenylate cyclase

Abstract

To investigate the effects of nucleoside triphosphates on the activation of adenylate cyclase by choleragen and on the stability and catalytic function of the choleragen-activated enzyme, samples of particulate preparation from bovine brain were successively treated in 3 separate incubations with extensive washing between each step. In incubation I, choleragen and NAD were present to activate the adenylate cyclase. In incubation II, conditions were varied to assess enzyme stability. Finally, adenylate cyclase activity was assayed with ATP or adenylyl imidodiphosphate as the substrate. Even when assays contained an optimal concentration of GTP, nucleoside triphosphate (plus a regenerating system) was required in incubation I for maximal choleragen activation; in order of effectiveness, GTP > ITP .mchgt. ATP .gtoreq. CTP = UTP. During incubation II (at 30.degree. C), activity of the choleragen-treated fractions was essentially completely stable when 100 .mu.M GTP (plus a regenerating system) was present. ITP and ATP were less effective. Activation produced by guanylyl imidodiphosphate was more stable than that resulting from choleragen, GTP and NAD. After activation of membranes with choleragen, NAD and GTP, nucleoside triphosphate plus a regenerating system (but not NAD or additional choleragen) was essential for expression of maximal activity. In order of effectiveness, GTP > ITP .mchgt. ATP .gtoreq. CTP = UTP. Thus GTP, which was effective in micromolar concentrations, plays an important role in the activation of adenylate cyclase by choleragen and in the stabilization and expression of the catalytic function of the activated enzyme.