Developmental regulation of the cyclic‐nucleotide‐phosphodiesterase mRNA of Dictyostelium discoideum

Abstract

Extracellular cyclic-nucleotide phosphodiesterase of D. discoideum has previously been purified and characterized. Antisera have been raised against the purified enyzme. Following cell-free translation of RNA extracted from cells at various stages of development and immunoprecipitation with anti-phosphodiesterase serum, cAMP phosphodiesterase synthesized in vitro and labeled with L-[35S]methionine can be detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. The cell-free translation product is an MW-48,000 polypeptide and can be immunoprecipitated with antiserum raised against active MW-50,000 cAMP phosphodiesterase or antiserum raised against heat-denatured cAMP phosphodiesterase. Purified native cAMP phosphodiesterase blocks immunoprecipitation of the cAMP-phosphodiesterase polypeptide synthesized in vitro. A detectable level of cAMP-phosphodiesterase mRNA is present in axenically grown cells. After starvation of the cells in phosphate buffer for 1 h an increase in translatable cAMP-phosphodiesterase mRNA occurs, followed by a decrease and another increase. When cells are starved in the presence of the slowly hydrolyzed cAMP analog, adenosine 3'',5''-thiophosphate, the level of translatable cAMP-phosphodiesterase mRNA increases about tenfold and does not show a temporary decline. A maximum of 0.015% of the total acid-insoluble radioactivity is incorporated into the MW-48,000 cAMP-phosphodiesterase polypeptide.