Thrombin stimulation of platelets causes an increase in phosphatidylinositol 5‐phosphate revealed by mass assay

FEBS Letters

Morris, James B¹  Irvine, Robin F¹  Ciruela, Antonio¹  Hinchliffe, Katherine A¹  Letcher, Andrew J¹ 

[1]Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK

关键词: Phosphatidylinositol 5-phosphate;    Platelet;    Thrombin;    Mass assay;    Type II phosphatidylinositol phosphate kinase;    Acid inhibition;   

DOI  :  10.1016/S0014-5793(00)01625-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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-³²P]ATP and recombinant PIPkin IIα, and quantifying the radiolabelled PtdInsP₂ 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.

【 授权许可】

Unknown