Mandon E C, Ehses I, Rother J, van Echten G, Sandhoff K
Institut für Organische Chemie und Biochemie, Universität Bonn, Germany.
J Biol Chem. 1992 Jun 5;267(16):11144-8.
Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.
丝氨酸棕榈酰转移酶、3-脱氢鞘氨醇还原酶和鞘氨醇N-酰基转移酶分别负责鞘脂生物合成的第一步，形成3-氧代鞘氨醇、鞘氨醇和二氢神经酰胺。我们使用高度纯化的小鼠肝脏内质网（ER）和高尔基体制剂，证实了这些酶在内质网中的定位。在甘露糖-6-磷酸酶潜伏率为90%的条件下，用不同的蛋白水解酶对密封的“外翻”小鼠肝脏内质网衍生囊泡进行温和消化，导致丝氨酸棕榈酰转移酶、3-脱氢鞘氨醇还原酶和鞘氨醇N-酰基转移酶活性失活约60-80%。这些鞘脂生物合成活性（丝氨酸棕榈酰转移酶、3-脱氢鞘氨醇还原酶和鞘氨醇N-酰基转移酶）不是潜伏性的，表明它们面向内质网的胞质侧，因此底物可以自由进入其活性位点。此外，与大量内质网蛋白结合的膜不可渗透化合物4,4'-二异硫氰酸芪-2,2'-二磺酸，可使丝氨酸棕榈酰转移酶和鞘氨醇N-酰基转移酶活性抑制30-70%。
