title: Evidence for cAMP-dependent protein kinase in the dinoflagellate, Amphidinium operculatum.
abstract: A cAMP dependent protein kinase (PKA) was identified in the dinoflagellate Amphidinium operculum. In vitro kinase activity towards kemptide, a PKA-specific substrate, was not detectable in crude lysates. However, fractionation of dinoflagellate extracts by gel filtration chromatography showed PKA-like activity toward kemptide at approximately 66 kDa. These findings suggest that possible low molecular mass inhibitors in crude lysates were removed by the gel filtration chromatography. Pre-incubation of extracts with cAMP prior to chromatography resulted in an apparent molecular mass shift in the in vitro kinase assay to 40 kDa. An in-gel kinase assay reflected activity of the free catalytic subunit at approximately 40 kDa. Furthermore, western blotting with an antibody to the human PKA catalytic subunit confirmed a catalytic subunit with a mass of approximately 40 kDa. Results from this study indicate that the PKA in A. operculatum has a catalytic subunit of similar size to that in higher eukaryotes, but with a holoenzyme of a size suggesting a dimeric, rather than tetrameric structure.
authors.affiliation: Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, NOAA, National Ocean Service, Charleston, South Carolina 29412, USA. tod.leighfield@noaa.gov
authors.forename: Tod A
authors.initials: TA
authors.lastname: Leighfield
authors.forename: Michèle
authors.initials: M
authors.lastname: Barbier
authors.forename: Frances M
authors.initials: FM
authors.lastname: Van Dolah
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004141:Dinoflagellida / Q000201:enzymology*; D007124:Immunoenzyme Techniques; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism
chemical_list: D009842:Oligopeptides; C037642:kemptide; D017868:Cyclic AMP-Dependent Protein Kinases