Protein kinase C activity and phosphoprotein pattern in stimulated alveolar macrophages.
Bovine alveolar macrophages (BAM) were stimulated with quartz dusts, metal oxide-coated silica particles, and zymosan. To investigate the role of protein kinase C (PKC) in the mechanism of agonist-induced activation 12-O-tetradecanoyl phorbol 13-acetate (TPA), staurosporine, and the PKC specific inhibitor GF 109203X were applied. PKC activity was determined by means of a continuous fluorescence assay [1]. The assay is based on the measurement of fluorescence decrease caused by phosphorylation of an acrylodan-labelled MARCKS peptide, a specific substrate of PKC. The PKC fluorescence assay was verified with the purified enzyme, but it could not be adapted to cytosolic and membrane homogenates of BAM, as it is sensitive to the activity of proteases. PKC-mediated protein phosphorylation in intact BAM was achieved by mapping the [32P]phosphoproteins with an optimized horizontal 2D electrophoresis technique with subsequent autoradiography and image analysis. Agonist- and time-dependent changes of phosphoprotein patterns in BAM were detected and analysed.
Radloff
M
M
Department of Biochemistry and Molecular Biology, University of Hamburg, Germany.
Gercken
G
G
D000818:Animals; D002417:Cattle; D003600:Cytosol / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D008262:Macrophage Activation / Q000502:physiology*; D016676:Macrophages, Alveolar / Q000737:chemistry / Q000201:enzymology*; D008301:Maleimides / Q000494:pharmacology; D010750:Phosphoproteins / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007211:Indoles; D008301:Maleimides; D010750:Phosphoproteins; D011493:Protein Kinase C; D019311:Staurosporine; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate