Kannan Muthukumar, Davis Deanna L, Suemitsu John, Oltorik Christopher D, Wattenberg Binks
Dept. of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA.
Bio Protoc. 2020 Jun 20;10(12):e3656. doi: 10.21769/BioProtoc.3656.
Serine palmitoyltranferase (SPT) is a pyridoxal 5' phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane proteins termed the ORMDLs. Here we describe a procedure to assay ORMDL dependent lipid inhibition of SPT activity. The assay of SPT activity using radiolabeled L-serine was developed from the procedure established by the Hornemann laboratory. The activity of SPT can also be measured using deuterated L-serine but it requires mass spectrometry, which consumes money, time and instrumentation. The ORMDL dependent lipid inhibition of SPT activity can be studied in both cells and in a cell free system. This assay procedure is applicable to any type of mammalian cell. Here we provide the detailed protocol to measure SPT activity in the presence of either short chain (C8-ceramide) or long chain ceramide (C24-ceramide). One of the greatest advantages of this protocol is the ability to test insoluble long chain ceramides. We accomplished this by generating long chain ceramide through endogenous ceramide synthase by providing exogenous sphingosine and 24:1 acyl CoA in HeLa cell membranes. This SPT assay procedure is simple and easy to perform and does not require sophisticated instruments.
丝氨酸棕榈酰转移酶（SPT）是一种依赖于磷酸吡哆醛（PLP）的酶，催化鞘脂合成的第一步也是限速步骤。SPT活性会根据鞘脂水平的升高进行稳态调节。SPT的这种稳态调节是通过称为ORMDL的小内质网（ER）膜蛋白介导的。在此，我们描述了一种检测ORMDL依赖性脂质对SPT活性抑制作用的方法。使用放射性标记的L-丝氨酸检测SPT活性的方法是在Hornemann实验室建立的方法基础上发展而来的。也可以使用氘代L-丝氨酸来测量SPT活性，但这需要质谱分析，既费钱又耗时，还需要仪器设备。ORMDL依赖性脂质对SPT活性的抑制作用可以在细胞和无细胞系统中进行研究。该检测方法适用于任何类型的哺乳动物细胞。在此，我们提供了在存在短链（C8-神经酰胺）或长链神经酰胺（C24-神经酰胺）的情况下测量SPT活性的详细方案。该方案最大的优点之一是能够检测不溶性长链神经酰胺。我们通过在HeLa细胞膜中提供外源性鞘氨醇和24:1酰基辅酶A，利用内源性神经酰胺合酶生成长链神经酰胺来实现这一点。这种SPT检测方法简单易行，不需要复杂的仪器。
