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

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Abstract
After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cAMP-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 labeling of the regulatory subunits of cAMP-dependent protein kinase with 8-azido-c[32P]AMP indicated that platelet lysate contained a 4-fold excess of 49,000-Da [dalton] 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 cAMP-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 cAMP-dependent activity was normally observed in the soluble fraction. The particulate cAMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (wt/vol) 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 cAMP-dependent holoenzyme and free RI subunits. Platelets contain 3 main protein kinase activities detectable with histone substrates, i.e., a membrane-bound type-I cAMP-dependent enzyme, a soluble type-II cAMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.

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