SUBUNIT STRUCTURE AND CATALYTIC PROPERTIES OF BOVINE BRAIN CA2+-DEPENDENT CYCLIC NUCLEOTIDE PHOSPHODIESTERASE

Ca2+-dependent cAMP phosphodiesterase has been purified 3000-fold to apparent electrophoretic and chromatographic homogeneity by affinity chromatography on Ca2+-dependent regulatory protein coupled to Sepharose followed by gel filtration on Sephadex G-200 and chromatography on hydroxylapatite. The purified enzyme contains three subunits of molecular weights of 61 000, 59 000, and 15 000. The 59 000 molecular weight polypeptide alone is associated with catalytic activity toward cAMP and cGMP as well as Ca2+ stimulation. The other two subunits appear to be those of the inhibitory protein previously described in this laboratory which seems to physically interact with the enzyme. This purified enzyme displays the catalytic properties characteristic of the “high Km” enzyme; it obeys Michael-is-Menten kinetics in the presence of Ca2+ and CDR. The Ca2+-dependent regulatory protein is shown to increase the Vm value by eight- to tenfold and to decrease the Km value two-to threefold under optimal conditions. The enzyme is also stimulated by NH4C1 which increases the Km and the Vm of the enzyme for cAMP both in the presence and absence of Ca2+-dependent regulator. Imidazole, another activator of the enzyme at high cAMP concentrations, increases Vm and Km for cAMP and inhibits the interaction of the enzyme with the Ca2+-dependent regulatory protein and can thereby become an inhibitor at 10“6 M cAMP and nonsaturating concentrations of CDR. © 1979, American Chemical Society. All rights reserved.