Divalent cation binding properties of bovine brain Ca2+-dependent regulator protein.

The divalent cation binding properties of a protein which functions as a Ca2+-dependent regulator (CDR) of brain cyclic nucleotide phosphodiesterase and adenylate cyclase activities have been examined by equilibrium dialysis and circular dichroic spectrophotometry. The far-ultraviolet circular dichroic spectrum is characterized by negative maxima at 207 and 222 nm. Changes in the far-ultraviolet circular dichroic spectrum, consistent with a coil to helix transition, occur upon addition of Ca2’, Mg2+, or Mn*+ to divalent cation-free CDR. The extent of the transition, however, at saturating concentrations of Ca*+ is greater than that observed at saturating concentrations of Mn2+ or Mg*+. The addition of Ca2+, Mg2+, or Mn 2+ likewise produced dramatic changes in the near-ultraviolet circular dichroic spectrum of CDR. The difference spectrum between divalent cation-free and divalent cation-containing conditions revealed that marked changes in the asymmetry of the microenvironment of both phenylalanine and tyrosine residues of CDR accompanied the binding of divalent cations. Binding studies utilizing the technique of equilibrium dialysis established the existence of two classes of Ca2+binding sites: sites class A with a capacity of 3 mol/mol of CDR and a dissociation constant of 2 x 10m7 M and site class B with a capacity of 1 mol/mol of CDR and a dissociation constant of 10e6 M. Mn’+ also bound to two classes of sites, 3 mollmol of CDR with a dissociation constant of 1.2 x lo-” M and 1 mol/mol of CDR with a dissociation constant of 4 x lo-” M. The identity of the Ca2+ and Mn*+ sites was established by competitive binding experiments. Mg”+ binding occurred at two classes of sites, 3 mol/mol of CDR with a dissociation constant of 1.4 x 10m4 M and 1 mol/mol of CDR with a dissociation constant of 2 x 10m5 M. The identity of the Ca2+ and Mg2+ binding sites was established by competitive binding experiments. At 1 mM Mg2+, binding of Ca2+ and Mn”+ to CDR occurred at a single class of sites, 3 mol/mol of CDR with apparent dissociation constants of 3 x lo-” M and 2 X 10e5 M, respectively. Cyclic nucleotide phosphodiesterase assayed at 1 mM Mg2+ and either 8 FM Ca*+ or 50 PM MS+ (concentrations sufficient to produce equal degrees of exchange of Ca2+ or Mn2+ for Mg2+ at sites class A) was activated by IO-fold lower concentrations of the Ca2+ than of the Mn2+. CDR complex. The incongruity of these species of CDR was further con-