Characterization of the protein kinase activities of human platelet supernatant and particulate fractions

• Published in: Biochemical journal 1984-03, Vol.218 (2), p.285-294
• Main Authors: Salama, S E, Haslam, R J
• Format: Article
• Language: English
• Subjects: Blood Platelets - drug effects; Blood Platelets - enzymology; chromatography; Chromatography, DEAE-Cellulose; Cyclic AMP - pharmacology; Electrophoresis, Polyacrylamide Gel; Humans; man; Octoxynol; photoaffinity labelling; platelets; Polyethylene Glycols - pharmacology; Protamine Kinase - blood; protein kinase; Protein Kinases - blood; Subcellular Fractions - drug effects; Subcellular Fractions - enzymology
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2180285

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.