Open AccessDevelopmental regulation of the cyclic‐nucleotide‐phosphodiesterase mRNA of Dictyostelium discoideum
Author(s) -
MULLENS Isabel A.,
FRANKE Jakob,
KAPPES Dietmar J.,
KESSIN Richard H.
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb08302.x
Subject(s) - dictyostelium discoideum , phosphodiesterase , immunoprecipitation , antiserum , cyclic nucleotide phosphodiesterase , biochemistry , biology , microbiology and biotechnology , gel electrophoresis , messenger rna , enzyme , gene , antibody , immunology
Extracellular cyclic‐nucleotide phosphodiesterase of Dictyostelium discoideum has previously been purified and characterized [Orlow et al. (1981) J. Biol. Chem. 256 , 7620–7627]. Antisera have been raised against the purified enzyme. 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 ‐[ 35 S]methionine can be detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. The cell‐free translation product is an M r ‐48000 polypeptide and can be immunoprecipitated with antiserum raised against active M r ‐50000 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 of translatable cAMP‐phosphodiesterase mRNA occurs, followed by a decrease and another increase. When cells are starved in the presence of the slowly hydrolyzed cAMP analogue, 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 M r ‐48000 cAMP‐phosphodiesterase polypeptide.