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Preparation of high specific activity tritium‐labelled leukotriene B4 suitable for radioligand binding assay
Authors:Stanislav I. Schramm  Igor Yu. Nagaev  Alan Sabirsh  Valeriy P. Shevchenko  Anastasiya S. Arkhipova  Jesper Z. Haeggström  Nikolay F. Myasoedov
Affiliation:1. Department of Chemistry of Physiologically Active Compounds, Institute of Molecular Genetics of the Russian Academy of Sciences, Kurchatov Sq. 2, Moscow 123182, Russia;2. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S‐171 77 Stockholm, Sweden
Abstract:
We describe a method of preparation of high specific activity tritium‐labelled leukotriene (LT) B4 from [5,6,8,9,11,12,14,15‐3H] arachidonic acid (AA; 6.66 TBq/mmol) utilizing a LTB4‐synthesizing enzyme system from rat basophilic leukemia (RBL‐1) cells. It was shown that both cyclooxygenase inhibitor indomethacin and adenosine 5′‐triphosphate induced [3H] AA transformation to [3H] LTB4. In optimized conditions up to 15% of total radioactivity of the incubation mixture was present in [3H] LTB4. A separation of [3H] LTB4 from other labelled C20:4 products was achieved by a three‐step reverse phase‐high‐performance liquid chromatography in methanol‐ and acetonitrile‐based solvent systems. [3H] LTB4 was confirmed to be identical to the naturally occurring LTB4 by a radioligand binding assay using a culture of HF1 cells that express a BLT1 receptor. Copyright © 2008 John Wiley & Sons, Ltd.
Keywords:[3H] leukotriene B4  enzymatic synthesis  rat basophilic leukemia (RBL‐1) cells  BLT1 receptor  radioligand binding assay
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