Lahiri Sujoy, Lee Hyunmi, Mesicek Judith, Fuks Zvi, Haimovitz-Friedman Adriana, Kolesnick Richard N, Futerman Anthony H
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
FEBS Lett. 2007 Nov 13;581(27):5289-94. doi: 10.1016/j.febslet.2007.10.018. Epub 2007 Oct 23.
Ceramide is a key metabolite in the pathway of sphingolipid biosynthesis. In mammals, ceramide is synthesized by N-acylation of a sphingoid long-chain base by a family of ceramide synthases (CerS), each of which displays a high specificity towards acyl CoAs of different chain lengths. We now optimize a previously-described assay for measuring CerS activity for use upon over-expression of mammalian CerS, and using these conditions, establish the K(m) value of each CerS towards sphinganine. Remarkably, the K(m) values towards sphinganine are all similar, ranging from 2 to 5microM, even for CerS proteins that are able to use more than one acyl CoA for ceramide synthesis (i.e. CerS4). The availability of this assay will permit further accurate characterization of the kinetic parameters of mammalian CerS proteins.
神经酰胺是鞘脂生物合成途径中的关键代谢产物。在哺乳动物中，神经酰胺由一族神经酰胺合酶（CerS）通过鞘氨醇长链碱基的N-酰化作用合成，其中每种酶对不同链长的酰基辅酶A都表现出高度特异性。我们现在优化了一种先前描述的用于测量CerS活性的测定方法，以便在哺乳动物CerS过表达时使用，并利用这些条件确定每种CerS对鞘氨醇的K(m)值。值得注意的是，即使对于能够使用不止一种酰基辅酶A进行神经酰胺合成的CerS蛋白（即CerS4），其对鞘氨醇的K(m)值也都相似，范围为2至5微摩尔。该测定方法的可用性将允许进一步准确表征哺乳动物CerS蛋白的动力学参数。
