MODE OF INTERACTION BETWEEN FORSKOLIN AND MANGANESE ION IN ACTIVATING CATALYTIC UNIT OF ADENYLATE CYCLASE FROM RAT BRAIN

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Rat brain adenylate cyclase was solubilized with a combination of 0.7% sodium cholate and 0.6M ammonium sulfate, and fractionated by addition of solid ammonium sulfate. The precipitate at 35% ammonium sulfate saturation contained neither guanine nucleotide-binding regulatory protein (G protein) nor calmodulin, and was used as the catalytic unit of the enzyme system. This catalytic unit was activated synergistically by forskolin and Mn^<2+>. An apparent K_m value for Mg-adenosine triphosphate (ATP) of the catalytic unit was about 80 μM in the basal state, while it increased in concurrence with the increase in the enzyme activity when forskolin was added to the assay system. The increase in the K_m value depended on the forskolin concentration up to 1μM, above which the value converged on ca.200μM. Furthermore, activation of the catalytic unit by forskolin was more marked at higher concentration of Mg-ATP. On the other hand, Mg^<2+> suppressed the increase in the K_m value for Mg-ATP by forskolin, though the value in the basal state was not changed by Mn^<2+> alone. These findings indicate that the activation of the catalytic unit by forskolin is accompanied by the change in the affinity for Mg-ATP and Mn^<2+> modifies the change.
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