This work discusses the regulation of glycogen metabolism in Dictyostelium discoideum during its developmental cycle. Specifically, the possible cAMP dependent regulation of glycogen phosphorylase and glycogen synthase was examined. In other systems, cAMP can regulate at the level of the gene (procaryotes, CAP protein) or at the level of covalent, reversible modification of the enzyme activity (eucaryotes, cAMP-dependent protein kinase-cAMPdPK). In Dictyostelium, glycogen phosphorylase and glycogen synthase have each been found to occur in two forms; one regulated allosterically and the other independent of allosteric regulation. The regulation of the two forms of glycogen phosphorylase was examined in single-cell suspensions to which cAMP or one of several cAMP analogs were added to mimic differentiative conditions. The allosterically regulated form of glycogen phosphorylase, phosphorylase b, decreased in the presence of cAMP while a corresponding increase in phosphorylase a, the non-allosterically regulated form of glycogen phosphorylase, was observed over an 8 hr period in the same cultures. In the presence of cAMP analogs, a similar time course of regulation for the two forms of glycogen phosphorylase occurred but only 2’deoxy-cAMP gave an effect comparable to cAMP. Under these same conditions, northern blot analysis of three developmentally regulated mRNAs--PL3, D11, and D3--revealed that normal gene regulation was occurring. Under conditions where elevation of intracellular cAMP was inhibited, neither regulation of phosphorylase enzyme activity nor of the 3 genes was observed. This indicated that under these conditions intracellular elevation of cAMP was necessary for the observed effects on enzyme and gene activity. This requirement for intracellular cAMP may indicate the involvement of a cAMPdPK. The properties of a phosphorylase b kinase found in amoebal extracts are described. The kinase activity coeluted with the phosphorylase b activity on a DE·52 anion exchange column. Under the conditions described conversion of the phosphorylase b activity to the phosphorylase a activity was observed. However, an increase in molecular weight to 104 kd (as seen for purified phosphorylase a) was not observed. The characterization of a partially purified glycogen synthase I and its developmental regulation are described. Also described are in vitro attempts to convert the I form to the allosterically regulated, D form, under conditions conducive to phosphorylation. / Ph. D.

A developmentally regulated cyclic AMP-dependent protein kinase has been recently reported in Dictyostelium discoideum. This report describes some of the physical and kinetic properties of the cAMP-dependent holoenzyme and its subunits. Gel filtration data suggests a holoenzyme Mr of 170,000-190,000, and catalytic and regulatory subunit Mrs of 40,000 and 49,000, respectively. These molecular weight determinations are compatible with an R₂C₂ subunit arrangement of the holoenzyme. Kinase activity required the presence of Mg²⁺ but cAMP binding to the enzyme was not dependent on divalent metal ions. The pH optimum for kinase activity was 7.5; the cAMP binding activity was not affected over a pH range of 5.0-10.0. The holoenzyme and isolated regulatory subunit had identical cAMP Kds of 28 nM. Cyclic AMP was able to dissociate the subunits when analyzed by density gradient centrifugation. Histone VII-S activated the subunits in the absence of cAMP but did not produce their dissociation. In contrast to the gel filtration data, sedimentation values indicated a dimeric holoenzyme structure. Reassociation of the subunits in the absence of cAMP occurred rapidly and was not dependent upon a preincubation with MgATP. High NaCl and low pH depressed both the total kinase activity and the ability of the subunits to reassociate as determined by activity ratio. MgATP did not decrease the ability of the holoenzyme to bind cAMP, neither did the holoenzyme possess a high affinity MgATP binding site. By the use of microdissection techniques holoenzyme levels were determined in individuals at each stage of development and in each cell type during development. Kinase activity was low and non-cAMP dependent in early aggregates but increased and became cAMP-dependent in later aggregates. Maximum activity and cAMP-dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single-stage occurred during culmination when the activity in the stalks was approximately one-fourth that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor. / Ph. D. / incomplete_metadata