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Thrombin stimulation of platelets causes an increase in phosphatidylinositol 5‐phosphate revealed by mass assay
Author(s) -
Morris James B,
Hinchliffe Katherine A,
Ciruela Antonio,
Letcher Andrew J,
Irvine Robin F
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01625-2
Subject(s) - phosphatidylinositol , thrombin , chemistry , platelet , stimulation , inositol , phosphatidic acid , kinase , chromatography , inositol phosphate , biochemistry , substrate (aquarium) , enzyme , phospholipid , receptor , membrane , endocrinology , biology , ecology , immunology
Phosphatidylinositol 5‐phosphate (PtdIns5P), a novel inositol lipid, has been shown to be the major substrate for the type II PtdInsP kinases (PIPkins) [Rameh et al. (1997) Nature 390, 192–196]. A PtdInsP fraction was prepared from cell extracts by neomycin chromatography, using a protocol devised to eliminate the interaction of acidic solvents with plasticware, since this was found to inhibit the enzyme. The PtdIns5P in this fraction was measured by incubating with [γ‐ 32 P]ATP and recombinant PIPkin IIα, and quantifying the radiolabelled PtdInsP 2 formed. This assay was used on platelets to show that during 10 min stimulation with thrombin, the mass level of PtdIns5P increases, implying the existence of an agonist‐stimulated synthetic mechanism.