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Properties of the adenylate cyclase catalytic unit from caudate nucleus.

J. L. Bender,E. Neer

Published 1983 in Journal of Biological Chemistry

ABSTRACT

The solubilized catalytic unit (C) of adenylate cyclase from bovine caudate nucleus was separated from the component which mediates activation by guanine nucleotides (G/F) by the method of Strittmatter and Neer (Strittmatter, and Neer, E. J. (1980) Proc. Natl. Acad Sci U. S. A. 77,6344-6348). The separated catalytic unit is much more active in the presence of Mn2+ than when M&+ is the only divalent cation. The affinity of the catalytic unit for its substrate, Me. ATP, is the same with either cation. Free Mn2+ activates the enzyme at concentrations greater than that required to convert all the ATP to MnATP. Mn2+ stabilized the catalytic unit even in the absence of ATP. These observations are consistent with the idea that Mn2+ can interact with C both at the active site (with ATP) and at a separate divalent cation site. Occupancy of this site by Mn2+ does not block its activation by guanine nucleotide-activated G/F (G/F*). The separated catalytic unit from caudate nucleus can be activated by the plant diterpene, forskolin. Forskolin does not significantly affect the apparent K, of the enzyme for ATP. With forskolin, the catalytic unit is active even with M&+ as the divalent cation. However, activation by forskolin is synergistic with that of Mn2+ and free Mn2+ still activates the enzyme. Forskolin also potentiates the activation of C by G/F*, but this depends critically on the order in which activators are added to the catalytic unit. Forskolin must be added before the catalytic unit is activated by G/F*, otherwise there is no potentiation. The highly active state induced by forskolin and G/F* persists after the removal of forskolin and decays with a half-time of 20 min at 23 “C. This state can be further activated by Mn2+ so that a 1000-fold activation of the catalytic unit can be produced by forskolin, G/F*, and Mn2+. Forskolin had no effect on the rate of activation of the G/F unit by guanosine 5’-@,y-imino)triphosphate or on the amount of G/F* needed to activate C. G/F is known to mediate the activation of adenylate cyclase by hormone receptors. The activity of the catalytic unit has been considered to reflect passively the activity state of the G/F unit. The studies with forskolin suggest that this may not always be the case and that the state of the catalytic

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