Sphingomyelin synthase activity measurement by the fluorescent product in cell culture medium or animal plasma

利用细胞培养基或动物血浆中的荧光标记产物测定鞘磷脂合酶活性(英文)

Sphingolipids, a new class of lipid mediators, are involved in a variety of important physiological and pathological processes. Sphingomyelin synthase （SMS） is an enzyme to convert the ceramide （Cer.） and phosphatidylcholine into sphingomyelin （SM） and diacylglycerol, which plays a key role in sphingolipid biosynthesis. Two SMS isoforms, SMS1 and SMS2, have been identified with different subceUular localizations and expression level in tissues. Previous studies have shown that SMS may serve as a potential therapeutic target for the treatment of various diseases, such as cardiovascular and metabolic diseases. Thus, there is an urgent need for a rapid and sensitive method for SMS activity analysis. In our study, we developed a novel method for SMS activity by monitoring the appearance of the product, NBD-SM, in the tissue culture medium or blood and applied this method in cells and mice. In Huh7 cells, the interassay coefficient of variation of the SMS activity assay was （3.60±0.07）% . In wild type （WT） mice, we observed accumulation of NBD-SM in blood in a time dependent fashion. In SMS2 KO mice, NBD-SM in plasma collected at 5- （0%, P〈0.01）, 30- （16%, P〈0.01）, and 60 min （21%, P〈0.01） after injection of fluorescence liposome solution was significantly decreased compared with WT mice. However, in SMS1 KO mice, NBD-SM in plasma collected 5- and 30 min is similar to that in WT mice. Our results suggest that this method could be used for SMS activity measurement in vitro and in vivo.

鞘磷脂合酶(SMS)催化神经酰胺(ceramide,Cer.)转变为鞘磷脂(sphingomyelin,SM)。近来的研究表明神经酰胺和鞘磷脂参与了代谢综合征的过程,因而鞘磷脂合酶被认为是开发抗代谢综合征药物的潜在靶点。鞘磷脂合酶有两个同工酶,分别称为鞘磷脂合酶1(SMS1)和鞘磷脂合酶2(SMS2)。这两种同工酶的亚细胞定位不同,在不同组织中的表达水平也有差异。到目前为止,已发表有多种方法测定组织和细胞匀浆中的总SMS活性,这些方法通过分析总反应体系或细胞内的酶促反应产物来衡量SMS活性。本文介绍一种测定SMS活性或筛选SMS抑制剂的新方法。我们将荧光标记的神经酰胺(NBDCer.)作为底物与细胞孵育,或者将该底物注射到小鼠体内,然后监测在细胞培养基或小鼠血浆中出现的荧光标记的鞘磷脂(NBDSM)的含量。采用这种办法可有效检测出D609(一种鞘磷脂合酶抑制剂)对细胞和小鼠整体SMS活性的抑制作用。我们进一步采用该方法检测了SMS1基因敲除小鼠和SMS2基因敲除小鼠的SMS活性,结果发现注射底物后,SMS2基因敲除小鼠(而不是SMS1基因敲除小鼠)血浆中NBDSM的堆积被明显阻断。因而该方法可用于检测生理或药理条件下在体或离体组织的SMS活性、筛选SMS抑制剂、甚至筛选SMS2特异性抑制剂。