A novel assay for measuring recombinant human lysophosphatidylcholine acyltransferase 3 activity
2019
Xinming Du | Jiachun Hu | Qing Zhang | Qi Liu | Xinxin Xiang | Jibin Dong | Bin Lou | Shuhua He | Xiang Gu | Yu Cao | Yingxia Li | Tingbo Ding
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.
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