Purification and characterization of an inhibitor protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase.

A heat-labile inhibitor protein of adenylate cyclase (EC 4.6.1.1) and phosphodiesterase (EC 3.1.4.17) has been purified to apparent homogeneity from bovine brain cerebrum by a simple two-column procedure. The inhibitor exerts its effect on adenylate cyclase or phosphodiesterase by forming a complex with the Ca2+-dependent activator protein, thereby competing with the apoenzyme for the activator. The protein was estimated to have a molecular weight of 80,000 and a Stokes radius of 39 A by gel filtration. The inhibitor was resolved in a sodium dodecyl sulfate-polyacrylamide gel into two equal molar subunits, with molecular weights of 60,000 and 18,500. In the presence of the activator and Ca2+, the thermal stability of the inhibitor was increased, indicative of a new conformation. The effectiveness of the inhibitor varied considerably, depending on its sequence of addition to the reaction mixture relative to phosphodiesterase and the activator protein, presumably because the activator appeared to have a greater affinity for the inhibitor than for phosphodiesterase.