Farrer R G, Warden M P, Quarles R H
Myelin and Brain Development Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
J Neurochem. 1995 Oct;65(4):1865-73. doi: 10.1046/j.1471-4159.1995.65041865.x.
Brefeldin A (BFA) has been used extensively to study the intracellular transport and processing of proteins and sphingolipids because of its dramatic alteration of the structural and functional organization of the Golgi. We have examined the effect of BFA on the synthesis of galactosylceramide sulfate (SGalCer) and its immediate precursor galactosylceramide (GalCer) in an immortalized Schwann cell line (S16) to determine the intracellular sites of synthesis of these two related glycolipids. During a 6-h labeling period, a dose-dependent inhibition of [35S]sulfate incorporation into SGalCer was observed with 95% inhibition occurring at 0.5 microgram/ml BFA. Labeling of newly synthesized galactosphingolipids with [3H]-palmitic acid for 6 h in the presence of BFA resulted in increased incorporation of label into GalCer containing nonhydroxy fatty acids (NFA-GalCer) to 162% of control values, whereas labeling of GalCer containing 2-hydroxy fatty acids (HFA-GalCer) was reduced to 63% of control. After 24 h, these values were at 366 and 91%, respectively. These results indicate that at least some of the HFA-GalCer was initially synthesized at a location distal to the BFA block and separate from the site of NFA-GalCer synthesis. Examination of [3H]palmitic acid incorporation into free ceramides showed an increase of 133 and 161% for hydroxy and nonhydroxy fatty acid ceramides, respectively, in cells treated for 6 h with BFA in comparison with levels found in untreated control cells, indicating that BFA did not block fatty acid 2-hydroxylation or the formation of HFA ceramide.(ABSTRACT TRUNCATED AT 250 WORDS)
布雷菲德菌素A（BFA）因其能显著改变高尔基体的结构和功能组织，已被广泛用于研究蛋白质和鞘脂的细胞内运输及加工过程。我们研究了BFA对永生化雪旺氏细胞系（S16）中硫酸半乳糖神经酰胺（SGalCer）及其直接前体半乳糖神经酰胺（GalCer）合成的影响，以确定这两种相关糖脂的细胞内合成位点。在6小时的标记期内，观察到[35S]硫酸盐掺入SGalCer呈剂量依赖性抑制，在0.5微克/毫升BFA时抑制率达95%。在BFA存在下，用[3H] - 棕榈酸对新合成的半乳糖鞘脂进行6小时标记，导致标记物掺入含非羟基脂肪酸的GalCer（NFA - GalCer）增加至对照值的162%，而含2 - 羟基脂肪酸的GalCer（HFA - GalCer）标记减少至对照的63%。24小时后，这些值分别为366%和91%。这些结果表明，至少部分HFA - GalCer最初是在BFA作用阻断点远端且与NFA - GalCer合成位点分开的位置合成的。检测[3H]棕榈酸掺入游离神经酰胺的情况，发现与未处理的对照细胞相比，用BFA处理6小时的细胞中，羟基和非羟基脂肪酸神经酰胺分别增加了133%和161%，这表明BFA并未阻断脂肪酸2 - 羟基化或HFA神经酰胺的形成。（摘要截短至250字）
