Protein kinase C activity and phosphoprotein pattern in stimulated alveolar macrophages
| Autor: | M Radloff, Günther Gercken |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Indoles
In Vitro Techniques Biology Toxicology Maleimides chemistry.chemical_compound Cytosol Macrophages Alveolar medicine Animals Staurosporine Electrophoresis Gel Two-Dimensional Protein phosphorylation MARCKS Protein Kinase C Protein kinase C food and beverages General Medicine Macrophage Activation Phosphoproteins Rats Spectrometry Fluorescence Biochemistry chemistry Tetradecanoylphorbol Acetate Phosphoprotein Phorbol Phosphorylation Cattle medicine.drug |
| Zdroj: | Toxicology Letters. 88:139-145 |
| ISSN: | 0378-4274 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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