Du Xinming, Hu Jiachun, Zhang Qing, Liu Qi, Xiang Xinxin, Dong Jibin, Lou Bin, He Shuhua, Gu Xiang, Cao Yu, Li Yingxia, Ding Tingbo
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, China.
Department of Pharmacology and Biochemistry, School of Pharmacy, Fudan University, Shanghai, China.
FEBS Open Bio. 2019 Oct;9(10):1734-1743. doi: 10.1002/2211-5463.12712. Epub 2019 Aug 30.
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 K values for substrates NBD-labelled lysophosphatidylcholine and arachidonoyl CoA were 266.84 ± 3.65 and 11.03 ± 0.51 μmol·L , respectively, and the V was 39.76 ± 1.86 pmol·min ·U . Moreover, we used our new method to determine the IC of a known LPCAT inhibitor, TSI-10. In conclusion, this novel assay can be used to measure the effects of compounds on LPCAT3 activity.
溶血磷脂酰胆碱酰基转移酶3（LPCAT3）是磷脂重塑过程中的一种重要酶，该过程会影响细胞膜的生物物理特性，进而影响细胞功能。多项证据表明，LPCAT3与多种疾病有关，包括动脉粥样硬化、非酒精性脂肪性肝炎和癌症。因此，LPCAT3可能具有作为这些疾病治疗靶点的潜力。在本研究中，我们设计了一种基于反相高效液相色谱的测定方法来测量LPCAT3活性，这可能有助于鉴定LPCAT3抑制剂和激活剂。我们发现重组人LPCAT3的最佳pH值和温度分别为6.0和30℃。底物NBD标记的溶血磷脂酰胆碱和花生四烯酰辅酶A的酶K值分别为266.84±3.65和11.03±0.51μmol·L，V为39.76±1.86 pmol·min·U。此外，我们使用新方法测定了已知LPCAT抑制剂TSI-10的IC。总之，这种新的测定方法可用于测量化合物对LPCAT3活性的影响。
