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Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum

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Summary

We have recently reported the existence of two forms of glycogen phosphorylase (1,4-α-D-glucan: orthophosphate-α-glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum. During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased. The total phosphorylase activity remained constant. The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme. In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK). We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5′AMP and a 104kd peptide appears. We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions. The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK. We found that relatively low levels, 100 μM, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity. The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK. Two other analogs, Bt2-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 μM) for ‘b’ to ‘a’ conversion. cDNAs to three cAMP-regulated genes-PL3, Dll, and D3-were used as controls in the above experiments. In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture - a medium in which adenylate cyclase is inhibited. Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP. The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.

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Abbreviations

EGTA:

Ethyleneglycol-bis-(β-aminoethyl ether)

N,N,N′:

N′-tetra acetic acid

SDS:

Sodium Dodecyl Sulfate

PAGE:

Polyacrylamide Gel Electrophoresis

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Author notes

  1. Charles L. Rutherford

    Present address: Biology Department, Virginia Tech University, 2031 Derring Hall, 24061, Blacksburg, VA, USA

Authors and Affiliations

  1. Department of Biology, Virginia Tech University, 24061, Blacksburg, VA, USA

    Debra A. Brickey, Venil Naranan, Joseph F. Sucic & Charles L. Rutherford

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  2. Venil Naranan
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  3. Joseph F. Sucic
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Brickey, D.A., Naranan, V., Sucic, J.F. et al. Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum . Mol Cell Biochem 97, 17–33 (1990). https://doi.org/10.1007/BF00231698

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  • DOI: https://doi.org/10.1007/BF00231698

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