Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.
Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.
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小鼠肝脏中丝氨酸棕榈酰转移酶、3-脱氢二氢鞘氨醇还原酶和二氢鞘氨醇 N-酰基转移酶的亚细胞定位和膜拓扑。
DOI：
10.1016/s0021-9258(19)49887-6
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
K. Sandhoff
中科院分区：
文献类型：
--
作者：
E. Mandon;I. Ehses;J. Rother;G. Echten;K. Sandhoff
丝氨酸棕榈酰转移酶、3-脱氢二氢鞘氨醇还原酶和二氢鞘氨醇N-酰基转移酶分别负责鞘脂生物合成的第一步，形成3-氧代二氢鞘氨醇、二氢鞘氨醇和二氢神经酰胺。我们证实了这些酶的本地化内质网（ER）使用高度纯化的小鼠肝脏ER和高尔基体制剂。在甘露糖-6-磷酸酶的潜伏期为90%的条件下，用不同的蛋白水解酶温和消化密封的“右侧外”小鼠肝脏ER衍生的囊泡，产生丝氨酸棕榈酰转移酶、3-脱氢二氢鞘氨醇还原酶和二氢鞘氨醇N-酰基转移酶活性的约60-80%失活。这些鞘脂生物合成活性（丝氨酸棕榈酰转移酶，3-脱氢二氢鞘氨醇还原酶和二氢鞘氨醇N-酰基转移酶）不是潜伏的，表明它们面向ER的胞质侧，因此底物可以自由进入其活性位点。此外，与大量ER蛋白结合的膜不可渗透化合物4，4 ′-二异硫氰基芪-2，2 ′-二磺酸抑制丝氨酸棕榈酰转移酶和鞘氨醇N-酰基转移酶活性30- 70%。
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%.