论文部分内容阅读
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。
Sphingomyelin synthase (SMS) catalyzes the conversion of ceramide (Cer.) To sphingomyelin (SM). Recent studies have shown that ceramide and sphingomyelin are involved in the process of metabolic syndrome and therefore sphingomyelin synthase is considered as a potential target for the development of anti-metabolic syndrome drugs. Sphingomyelin synthase has two isozymes, called sphingomyelin synthase 1 (SMS1) and sphingomyelin synthase 2 (SMS2), respectively. The subcellular localization of these two isozymes is different and their expression levels are also different in different tissues. So far, various methods have been published for measuring total SMS activity in tissue and cell homogenates, which measure SMS activity by analyzing total reaction system or intracellular enzymatic reaction products. This article describes a new method for measuring SMS activity or screening SMS inhibitors. We incubated the cells with fluorescently labeled ceramide (NBDCer.) As a substrate or injected the mouse into vivo and then monitored fluorescently labeled sphingomyelin (NBDSM) that appeared in the cell culture medium or mouse plasma Content. This approach effectively detects the inhibitory effect of D609, a sphingomyelin synthase inhibitor, on overall SMS activity in cells and in mice. We further examined SMS activity in SMS1 knockout mice and SMS2 knockout mice by this method and found that NBDSM in the plasma of SMS2 knockout mice (but not SMS1 knockout mice) after injection of the substrate The accumulation is obviously blocked. Thus the method can be used to detect SMS activity in vivo or in vitro under physiological or pharmacological conditions, screen SMS inhibitors, and even screen SMS2-specific inhibitors.