A rapid assay for activity of phospholipase A2 using radioactive substrate
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Human peroxiredoxin 6 is essential for malaria parasites and provides a host-based drug target
2022, Cell ReportsCitation Excerpt :Recombinant human PRDX6 (150 μg/mL final) and active MAPK (ERK2, R&D Systems, 10 μg/mL final) were added to 30 μL of a phosphorylation buffer containing 50 mM Tris/HCl pH 7.5, 20 μM EGTA, 10 mM MgCl2 and 2 mM Mg-ATP (Sigma) and were incubated for 90 min at 30°C. Measurement of PLA2 activity of phosphorylated PRDX6 (pPRDX6) was based on the enzymatic PLA2 assay described by Wu et al. (Wu et al., 2009) and the rapid free fatty acid extraction method by Katsumata et al. (Katsumata et al., 1986). Liposomes consisting of DPPC/egg yolk PC/egg yolk PG/cholesterol (6:3:1.2:0.95, DPPC from Avanti Polar Lipids, all other Sigma) with 0.6 μCi tracer [2-palmitoyl-1-14C]-dipalmitoyl phosphatidylcholine (14C-DPPC, American Radiolabeled Chemicals) were prepared by freezing/thawing three times in liquid nitrogen.
Development of high-throughput screening assays for profiling snake venom phospholipase A<inf>2</inf> activity after chromatographic fractionation
2020, ToxiconCitation Excerpt :Assays using chromogenic molecules for PLA2 profiling have previously been developed (Petrovic et al., 2001; Sharko and Kisel, 2011). One of these assays relies on hydrolysis of a substrate by PLA2s into a coloured product, with ensuing absorbance measured at 425 nm, as described by Petrovic et al. (2001) In addition, both radioactivity based assays (Aufenanger et al., 1993; Katsumata et al., 1986) and fluorogenic assays (Darrow et al., 2011; Mitnaul et al., 2007) have been described. All these assay formats, however, are probe substrate dependent and thus dependent on the selectivity and enzymatic activity of each PLA2 for the probe substrate used.
Changes in group II phospholipase A<inf>2</inf> gene expression in rat heart during sepsis
2013, Journal of Surgical ResearchCitation Excerpt :Only those animals that survived at each designated time point were included in the experiments. PLA2 activity was assayed by the method of Katsumata et al. [13] with modification as previously described by us [5,14]. Briefly, heart tissues were homogenized and the homogenate was filtered and centrifuged at 700 g for 10 min to remove nuclei and cell debris.
Fast method for monitoring phospholipase A<inf>2</inf> activity by liquid chromatography-electrospray ionization mass spectrometry
2009, Journal of Chromatography AA facile synthesis of 1-ethoxy-4-cyano-5-ethoxycarbonyl-3H-azuleno[1,2-c] pyran-3-one, a selective 15-lipoxygenase inhibitor
2004, Bioorganic and Medicinal Chemistry LettersHigh-performance liquid chromatographic assay with ultraviolet spectrometric detection for the evaluation of inhibitors of secretory phospholipase A<inf>2</inf>
2003, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences