HPLC assay with UV spectrometric detection for the evaluation of inhibitors of cytosolic phospholipase A2.
A non-radioactive spectrometric assay for the evaluation of inhibitors of cytosolic phospholipase A(2) (cPLA(2)) is described. The enzyme was isolated from human platelets applying anion exchange chromatography. Sonicated covesicles consisting of 0.2mM 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 0.1mM 1,2-dioleoyl-sn-glycerol were used as enzyme substrate. The cPLA(2) activity was determined by measuring the arachidonic acid released by the enzyme with reversed-phase HPLC and UV detection at 200 nm after cleaning up the samples by solid phase extraction. Two known cPLA(2) inhibitors were used to validate the test assay.
Schmitt
Melanie
M
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Hittorfstrasse 58-62, D-48149 Münster, Germany.
Lehr
Matthias
M
D016718:Arachidonic Acid / Q000737:chemistry; D001792:Blood Platelets / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003600:Cytosol / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000032:analysis*; D006801:Humans; D015394:Molecular Structure; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D054467:Phospholipases A2; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2