title: Endogenous substrates for in vitro phosphorylation by cyclic AMP-dependent protein kinase from Dictyostelium discoideum.
abstract: Endogenous proteins which could serve as substrates for cyclic AMP-dependent protein kinase in vitro were measured in cytosolic fractions at four stages of development. A peak of cyclic AMP-dependent phosphorylation occurred at the slug stage, coincident with the appearance of cyclic AMP-dependent protein kinase. After partial purification of the slug-stage extracts by DE-52 cellulose and Sephacryl S-300 chromatography, cyclic AMP dependency of six proteins was observed. The apparent subunit molecular weights of the proteins were greater than 200,000, 110,000, 107,000, 91,000, 75,000 and 69,000. Upon further purification of the cyclic AMP-dependent protein kinase by chromatofocusing, the endogenous substrates were separated from the enzyme. In addition, the enzyme separated into catalytic and regulatory subunits. If the purified catalytic subunit was added to heated S300 fractions, proteins with apparent molecular weights of 91,000 and 107,000 were specificity phosphorylated. The results show the stage-dependent appearance of a cyclic AMP-dependent protein kinase and point out several in vitro substrates for the enzyme.
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004023:Dictyostelium / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D005656:Fungal Proteins / Q000378:metabolism*; D007525:Isoelectric Focusing; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D011494:Protein Kinases