Marggraf W D, Zertani R, Anderer F A, Kanfer J N
Biochim Biophys Acta. 1982 Mar 12;710(3):314-23. doi: 10.1016/0005-2760(82)90114-x.
The intracellular location of sphingomyelin formation via the cholinephosphotransferase reaction from both endogenous an exogenous phosphatidylcholine and ceramide substrates has been studied in the subcellular membrane fractions prepared from mouse fibroblasts. The enzyme was found to be located in both the plasma membrane and the Golgi fractions. Activity in the Golgi fraction was stimulated to a greater extent by the addition of exogenous ceramide than was the activity in the plasma membrane fraction. It is concluded that endogenous phosphatidylcholine is available to the cholinephosphotransferase at saturating concentration and, therefore, is not rate-limiting. In contrast, the very low concentration of endogenous ceramide seems to limit the reaction rate, necessitating supplementation with exogenous material Both endogenous substrates are shown to be utilized in an intramembranous rather than an intermembranous reaction. The capacity of the plasma membrane fraction to synthesize sphingomyelin from endogenous phosphatidylcholine and ceramide was found to be sufficiently high to account for the rate of net synthesis of plasma membrane-bound sphingomyelin observed in the logarithmically multiplying cell culture. In contrast, the Golgi fraction displayed only 26% of the expected capacity, but it was stimulated 6-fold by the addition of exogenous ceramide. These results demonstrate that the total cellular sphingomyelin of the mouse fibroblasts can be provided via the cholinephosphotransferase pathways and that the plasma membrane and the Golgi fraction are most probably the intracellular sites of sphingomyelin biosynthesis.
通过胆碱磷酸转移酶反应，利用内源性和外源性磷脂酰胆碱及神经酰胺底物生成鞘磷脂的细胞内定位，已在从小鼠成纤维细胞制备的亚细胞膜组分中进行了研究。发现该酶位于质膜和高尔基体组分中。与质膜组分中的活性相比，添加外源性神经酰胺对高尔基体组分中的活性刺激作用更大。得出的结论是，内源性磷脂酰胆碱在饱和浓度下可被胆碱磷酸转移酶利用，因此不是限速因素。相比之下，内源性神经酰胺的浓度极低，似乎限制了反应速率，因此需要补充外源性物质。两种内源性底物均显示在膜内而非膜间反应中被利用。发现质膜组分从内源性磷脂酰胆碱和神经酰胺合成鞘磷脂的能力足够高，足以解释在对数增殖的细胞培养物中观察到的质膜结合鞘磷脂的净合成速率。相比之下，高尔基体组分仅显示出预期能力的26%，但添加外源性神经酰胺后其活性被刺激了6倍。这些结果表明，小鼠成纤维细胞的总细胞鞘磷脂可通过胆碱磷酸转移酶途径提供，并且质膜和高尔基体组分很可能是鞘磷脂生物合成的细胞内位点。
