The proposed research project is directed toward the elucidation of the function of a Ca ion-binding protein of brain. Primary emphasis will involve an investigation of the Ca ion-binding protein as a modulator protein conferring Ca ion sensitivity on a number of strategic enzyme systems of brain, including those central to cyclic nucleotide metabolism, namely, adenylate cyclase and cyclic nucleotide phosphodiesterase. A form of each of these enzymes has been shown to be deactivated by the chromatographic removal on ECTEOLA-cellulose of an endogenous Ca ion-dependent regulator (CDR) protein identified subsequently as the Ca ion-binding protein whose function is the subject of this grant. The CDR-sensitive forms of these enzymes will be purified to apparent homogeneity and characterized physically and kinetically. The Ca ion-dependent interactions of the protomers constituting these enzymes, as well as the participation of Mg ion and substrate in these interactions, will be explored with respect to changes of tertiary structure by circular dichroic spectral analyses. These interactions will be characterized further by reaction kinetics. Antibodies prepared against the purufied enzymes will be used in immunohistochemical fluorescent localization studies. Various cloned lines of neuroblastoma and glioma will be examined for the presence of CDR, CDR-dependent cyclic nucleotide phosphodiesterase and CDR-dependent adenylate cyclase. The effects of a variety of neurohormones and glioma will be examined for the presence of CDR, CDR-dependent cyclic nucleotide phosphodiesterase and CDR-dependent adenylate cyclase. The effects of a variety of neurohormones and ionophores on Ca ion efflux and influx rates will be studied in these cells and correlated with effects on intracellular cAMP and cGMP concentrations. A variety of additional key regulatory enzymes will be examined for possible Ca ion sensitivity mediated by this Ca ion-binding protein in order to ascertain whether its effects are restricted to cyclic nucleotide metabolism or are more general, involving coupling of cell stimulation with resultant Ca ion-flux to cell response by activation of diverse enzyme systems.