Cyclic 3',5'-nucleotide phosphodiesterase. Evidence for and properties of a protein activator.

Purification of cyclic 3’,5’-nucleotide phosphodiesterase of bovine brain cerebrum resulted in partial loss of activity, due to dissociation of an activator (or cofactor) from the enzyme. A systematic study showed that as purification proceeded, the activator was removed from phosphodiesterase in a stepwise fashion. Fractions representing phosphodiesterase at different stages of purification contained different concentrations of the activator in relation to phosphodiesterase. The fractions obtained in the initial stages contained excess or sufficient activator relative to the enzyme, and they required no exogenous cofactor for optimal activity. The fraction obtained in the last stage of purification was deficient in the cofactor, and phosphodiesterase was relatively inactive unless supplemented with an exogenous activator. The activator was sensitive to proteolytic enzymes, but not to RNase or DNase, indicating that it was a protein. The protein was resistant to 8 M urea, and to boiling at pH 1.7. Boiling at pH 12.3 obliterated all activity. Gel filtration indicated that the protein had a molecular weight of 40,000. Kinetic analysis showed that stimulation was independent of the time of preliminary incubation with the activator, but dependent on its concentration in the reaction mixture. In contrast, stimulation of the enzyme by trypsin was a catalytic process. Although mechanistically different, both agents increased the V,,,ax of the enzyme and decreased its K, for adenosine 3’,5’-monophosphate. An activator and a relatively inactive phosphodiesterase were also isolated from human brain, porcine brain, and rat brain as well as from bovine heart. An activator from one tissue cross-activated effectively a purified phosphodiesterase from another tissue, indicating a lack of tissue specificity. However, the effect of the activator was specific; none of a variety of proteins mimicked its action. A heated purified preparation lost its enzymic activity, but was capable of stimulating another preparation which had not been heated. It was concluded that the residual activity associated with the pursed enzyme was due to an incomplete removal of the activator from the enzyme