Open AccessDetection and Regulation of the mRNA for the Inhibitor of Extracellular cAMP Phosphodiesterase of Dictyostellum discoideum
Author(s) -
ROSSIER Claude,
FRANKE Jak ob,
MULLENS Isabel A.,
KELLEY Kenneth J.,
KESSIN Richard H.
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07474.x
Subject(s) - dictyostelium discoideum , methionine , biochemistry , cysteine , messenger rna , immunoprecipitation , microbiology and biotechnology , extracellular , phosphodiesterase , in vitro , glycoprotein , gel electrophoresis , enzyme inhibitor , protein biosynthesis , polyacrylamide gel electrophoresis , enzyme , inhibitor protein , biology , amino acid , gene
The inhibitor of the cAMP phosphodiesterase of Dictyostelium discoideum is a cysteine‐rich glycoprotein, which binds to the enzyme and inactivates it. When the inhibitor is removed, enzymatic activity is restored. Following translation in vitro of RNA from developing cells and immunoprecipitation with anti‐inhibitor serum, newly synthesized inhibitor can be detected by sodium dodecylsulfate/polyacrylamide gel electrophoresis and fluorography. The inhibitor can be labeled using [ 35 S]cysteine but not [ 35 S]methionine, in agreement with the previously determined amino acid composition, and can be detected after cell‐free translation only if it has been previously acetylated. Purified native inhibitor blocks immunoprecipitation of the inhibotor polypeptide synthesized in vitro . No inhibitor mRNA was detected in growing cells. Translatable mRNA was present 2 h after the beginning of starvation, reached a maximal level after 3 h, and decreased thereafter. Addition of 1 mM cAMP at the beginning of starvation delayed the appearance of translatable inhibitor mRNA. In the presence of 5 μM adenosine cyclic‐3′,5′‐phosphorothioate, a slowly hydrolyzed cAMP analogue, no translatable mRNA could be detected. Following removal of the analogue, the mRNA appeared within one hour and inhibitor was secreted after another hour.