A novel assay for measuring recombinant human lysophosphatidylcholine acyltransferase 3 activity

FEBS Open Bio
Xinming DuTingbo Ding

Abstract

Lysophosphatidylcholine acyltransferase 3 (LPCAT3) is an important enzyme in phospholipid remodeling, a process that influences the biophysical properties of cell membranes and thus cell function. Multiple lines of evidence suggest that LPCAT3 is involved in several diseases, including atherosclerosis, non-alcoholic steatohepatitis, and carcinoma. Thus, LPCAT3 may have potential as a therapeutic target for these diseases. In the present study, we devised an assay based on reversed-phase HPLC to measure LPCAT3 activity, which may facilitate the identification of LPCAT3 inhibitors and activators. We found that optimal pH and temperature of recombinant human LPCAT3 are 6.0 and 30 °C, respectively. The enzyme Km values for substrates NBD-labelled lysophosphatidylcholine and arachidonoyl CoA were 266.84 ± 3.65 and 11.03 ± 0.51 μmol·L-1 , respectively, and the Vmax was 39.76 ± 1.86 pmol·min-1 ·U-1 . Moreover, we used our new method to determine the IC50 of a known LPCAT inhibitor, TSI-10. In conclusion, this novel assay can be used to measure the effects of compounds on LPCAT3 activity.

References

Jan 16, 2008·The Journal of Biological Chemistry·Yang ZhaoGuoqing Cao
Apr 9, 2009·Journal of Lipid Research·Shilpa JainPeter Oelkers
Dec 17, 2009·Journal of Inorganic Biochemistry·Zhi-Qiang WangDennis J Stuehr
Sep 15, 2010·Gene·O DemeureS Lagarrigue
May 24, 2013·Journal of Biochemistry·Hideo ShindouTakao Shimizu
Feb 25, 2014·Journal of Lipid Research·Sarah A MartinRobert C Murphy
May 23, 2014·Journal of Lipid Research·Megumi TaruiTakao Shimizu
Jul 7, 2017·Journal of Vascular Research·Hiroki TanakaNaoki Unno
Apr 21, 2018·Biochimica Et Biophysica Acta. Molecular and Cell Biology of Lipids·Chunyan FengTingbo Ding
Nov 1, 2018·Annual Review of Physiology·Bo Wang, Peter Tontonoz

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Methods Mentioned

BETA
protein assay
size exclusion chromatography

Software Mentioned

Excel
prism
GraphPad

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