1-Hexadecyl-2-Arachidonoylthio-2-deoxy-sn-Glycero-3-Phosphorylcholine as a Substrate for the Microtiterplate Assay of Human Cytosolic Phospholipase A2

doi:10.1006/abio.1994.1079

Analytical Biochemistry

Volume 217, Issue 1, 15 February 1994, Pages 25-32

https://doi.org/10.1006/abio.1994.1079 Get rights and content

Abstract

Human cytosolic phospholipase A₂ (cPLA₂) is an 85-kDa protein which displays a preference for arachidonoyl phospholipids as substrates. This substrate preference and the assay characteristics of the enzyme are quite different from those of the smaller, more well-studied extracellular PLA₂s. We now report the development of a nonradioactive, spectrophotometric, microtiterplate assay for human cPLA₂ using a novel synthetic thio-phospholipid analog as a substrate. This substrate is a phosphatidylcholine derivitive with an arachidonoylthioester in the sn-2 position and an alkyl-ether in the sn-1 position. The use of an sn-1 alkyl-ether in the substrate ensures that the assay will only measure PLA₂ activity and will not be complicated by the metabolism of the lysophospholipid product by the enzyme′s lysophospholipase activity. cPLA₂ is assayed at pH 7.4 and 37°C with a mixed micellar substrate consisting of 2 mM thio-phospholipid and 4 mM Triton X-100 in 30% glycerol. Under these conditions, the assay is fairly linear for over 1 h.

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Primary and secondary hypertension is associated with kidney redox imbalance resulting in enhanced reactive oxygen species (ROS) and enzymes dependent phospholipid metabolism. The fatty acid amide hydrolase inhibitor, URB597, modulates the levels of endocannabinoids, particularly of anandamide, which is responsible for controlling blood pressure and regulating redox balance. Therefore, this study aimed to compare the effects of chronic URB597 administration to spontaneously hypertensive rats (SHR) and rats with secondary hypertension (DOCA-salt rats) on the kidney metabolism associated with the redox and endocannabinoid systems. It was shown fatty acid amide hydrolase (FAAH) inhibitor decreased the activity of ROS-generated enzymes what resulted in a reduction of ROS level. Moreover varied changes in antioxidant parameters were observed with tendency to improve antioxidant defense in SHR kidney. Moreover, URB597 administration to hypertensive rats decreased pro-inflammatory response, particularly in the kidneys of DOCA-salt hypertensive rats. URB597 had tendency to enhance ROS-dependent phospholipid oxidation, estimated by changes in neuroprostanes in the kidney of SHR and reactive aldehydes (4-hydroxynonenal and malondialdehyde) in DOCA-salt rats, in particular. The administration of FAAH inhibitor resulted in increased level of endocannabinoids in kidney of both groups of hypertensive rats led to enhanced expression of the cannabinoid receptors type 1 and 2 in SHR as well as vanilloid receptor 1 receptors in DOCA-salt rats. URB597 given to normotensive rats also affected kidney oxidative metabolism, resulting in enhanced level of neuroprostanes in Wistar Kyoto rats and reactive aldehydes in Wistar rats. Moreover, the level of endocannabinoids and cannabinoid receptors were significantly higher in both control groups of rats after URB597 administration.
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To detect cPLA2 activity arachidonoyl thio-PC as a substrate was used. Hydrolysis of the arachidonoyl thioester bond at the sn-2 position by PLA2 releases a free thiol which can be detected by DTNB [27]. Enzyme specific activity was calculated in nanomol of free thiol released per minute per milligram of protein.
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To detect phospholipase activity arachidonoyl thio-PC was used as a substrate. Hydrolysis of the arachidonoyl thioester bond at the sn-2 position by PLA2 releases a free thiol which is spectrophotometrically detected by reaction with DTNB at 405 nm (Reynolds et al., 1994). Western blot analysis of homogenates protein was performed according to Eissa and Seada (1998).
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Citation Excerpt :
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Regular Article1-Hexadecyl-2-Arachidonoylthio-2-deoxy-sn-Glycero-3-Phosphorylcholine as a Substrate for the Microtiterplate Assay of Human Cytosolic Phospholipase A2

Abstract

Regular Article
1-Hexadecyl-2-Arachidonoylthio-2-deoxy-sn-Glycero-3-Phosphorylcholine as a Substrate for the Microtiterplate Assay of Human Cytosolic Phospholipase A₂