Futerman A H, Stieger B, Hubbard A L, Pagano R E
Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210-3301.
J Biol Chem. 1990 May 25;265(15):8650-7.
The intracellular site of sphingomyelin (SM) synthesis was examined in subcellular fractions from rat liver using a radioactive ceramide analog N-([1-14C]hexanoyl)-D-erythro-sphingosine. This lipid readily transferred from a complex with bovine serum albumin to liver fractions without disrupting the membranes, and was metabolized to radioactive SM. To prevent degradation of the newly synthesized SM to ceramide, all experiments were performed in the presence of EDTA to minimize neutral sphingomyelinase activity and at neutral pH to minimize acid sphingomyelinase activity. An intact Golgi apparatus fraction gave an 85-98-fold enrichment of SM synthesis and a 58-83-fold enrichment of galactosyltransferase activity. Controlled trypsin digestion demonstrated that SM synthesis was localized to the lumen of intact Golgi apparatus vesicles. Although small amounts of SM synthesis were detected in plasma membrane and rough microsome fractions, after accounting for contamination by Golgi apparatus membranes, their combined activity contributed less than 13% of the total SM synthesis in rat liver. Subfractions of the Golgi apparatus were obtained and characterized by immunoblotting and biochemical assays using cis/medial (mannosidase II) and trans (sialyltransferase and galactosyltransferase) Golgi apparatus markers. The specific activity of SM synthesis was highest in enriched cis and medial fractions but far lower in a trans fraction. We conclude that SM synthesis in rat liver occurs predominantly in the cis and medial cisternae of the Golgi apparatus and not at the plasma membrane or endoplasmic reticulum as has been previously suggested.
利用放射性神经酰胺类似物N-([1-¹⁴C]己酰基)-D-赤型鞘氨醇，对来自大鼠肝脏的亚细胞组分中鞘磷脂（SM）的合成细胞内位点进行了检测。这种脂质很容易从与牛血清白蛋白形成的复合物转移至肝脏组分，而不会破坏膜结构，并且会代谢为放射性SM。为防止新合成的SM降解为神经酰胺，所有实验均在EDTA存在的条件下进行，以尽量降低中性鞘磷脂酶活性，并在中性pH条件下进行，以尽量降低酸性鞘磷脂酶活性。完整的高尔基体组分使SM合成富集了85 - 98倍，半乳糖基转移酶活性富集了58 - 83倍。可控的胰蛋白酶消化表明，SM合成定位于完整高尔基体囊泡的腔内。尽管在质膜和粗面微粒体组分中检测到少量的SM合成，但在考虑到被高尔基体膜污染的情况后，它们的综合活性在大鼠肝脏总SM合成中所占比例不到13%。通过使用顺面/中间（甘露糖苷酶II）和反面（唾液酸转移酶和半乳糖基转移酶）高尔基体标记物进行免疫印迹和生化分析，获得了高尔基体的亚组分并对其进行了表征。SM合成的比活性在富集的顺面和中间组分中最高，但在反面组分中则低得多。我们得出结论，大鼠肝脏中的SM合成主要发生在高尔基体的顺面和中间潴泡中，而不是如先前所认为的发生在质膜或内质网中。
