Cyclic AMP-binding proteins in human blood platelets detected by photoaffinity labelling

Chambers, Donald A.; Nachman, Ralph L.; Evarts, Joan; Kinoshita, T.

doi:10.1016/0304-4165(82)90090-3

Cited by 3 publications

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“…A similar peptide has been observed in brain (Lohmann et al, 1980). The failure of Chambers et al (1982) to detect RI, subunits in human platelets probably stems from the low concentration of 8-azido-cyclic [32P]AMP that they used (0.01-0.1 iM). Lyons (1980) has shown that, for unknown reasons, complete photoaffinity labelling of the platelet RI, subunit requires at least 0.4 uM reagent.…”

Section: Discussionmentioning

confidence: 99%

“…Two studies utilizing 8-azido-cyclic [32P]AMP as a photoaffinity label have shown that, when platelets were sonicated, the RI, regulatory subunits of platelet cyclic AMP-dependent protein kinase were found almost exclusively in the supernatant fraction, whereas at least half of the RI subunits were found in the particulate fraction (Lyons, 1980;Salama & Haslam, 1981a). However, a third study (Chambers et al, 1982) failed to detect RI, regulatory subunits in any subcellular fraction, though RI subunits were found in all platelet fractions. In the present work, we have sought to clarify the subcellular distribution of the isoenzymes of cyclic AMP-dependent protein kinases in human platelets, using both DEAE-cellulose chromatography and photoaffinity labelling.…”

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

Salama¹,

Haslam²

1984

Biochemical Journal

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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.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

Salama¹,

Haslam²

1984

Biochemical Journal

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Protein kinases in human leukemic cells

et al. 1985

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Protein kinase activities and cyclic AMP binding capacity were investigated in human peripheral blood cells from leukemic patients and normal controls. Using [gamma 32P] ATP as phosphoryldonor, the phosphorylating activities were not found to be significantly different in either normal or leukemic cells when measured on both artificial basic and acidic substrates. In contrast, the GTP-dependent casein kinase activity, CK2, which is almost undetectable in normal granulocytes, was markedly increased in highly proliferating myeloblastic cells from patients with acute myelogenous leukemia (AML) or with chronic myelogenous leukemia in blastic crisis (BC-CML). Levels of endogenous phosphotyrosine were not higher in leukemic cells than in normal peripheral lymphocytes or granulocytes. Finally, cAMP binding capacity was found to be increased in several types of proliferating leukemic cells, due to a higher amount of the R1-type regulatory subunit of the cAMP-dependent protein kinases. Specific patterns of cAMP binding proteins observed in the different types of normal blood cells were rather blurred in leukemic cells. In conclusion, modifications observed in human leukemic cells seem to be more related to proliferation or blockage in normal differentiation than to their cellular origin.

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Photoaffinity labeling of adenosine 3′: 5′ monophosphate binding proteins of human platelets

Fossett

Ganguly

1983

Thrombosis Research

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Cyclic AMP-binding proteins in human blood platelets detected by photoaffinity labelling

Cited by 3 publications

References 18 publications

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

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

Protein kinases in human leukemic cells

Photoaffinity labeling of adenosine 3′: 5′ monophosphate binding proteins of human platelets

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