Antes P, Schwarzmann G, Sandhoff K
Institut für Organische Chemie und Biochemie Universität, Bonn/Deutschland.
Eur J Cell Biol. 1992 Oct;59(1):27-36.
Fluorescent D-erythro-sphingosines bearing the diphenyl-1,3,5-hexatrienyl group (DPH) as fluorophore were synthesized for the first time. Two isomers, the DPH-4(E)- and DPH-4(Z)-sphingosine [(2S,3R)-2-amino-6-(p-(18-phenyl)-13,15,17(E,E,E)-hexatrienyl)phenylh ex- 4(E/Z)-en-1,3-diol], and the N-hexanoyl derivative of DPH-4(E)-sphingosine (C6-DPH-ceramide) were studied for their distribution and metabolism in cultured human skin fibroblasts. Both DPH-sphingosines (4-trans and 4-cis) were not significantly acylated to ceramide in living cells, but converted to ceramide in vitro by microsomal protein from mouse brain, although slower than natural D-erythro-sphingosine. DPH-4(Z)-sphingosine showed the same Km like D-erythro-sphingosine (155 microM), but had a lower Vmax value, 0.85 instead of 1.9 nmol/mgh. An even poorer substrate was DPH-4(E)-sphingosine with a Km of 220 microM and a Vmax of 0.81 nmol/mgh. In cultured human fibroblasts, C6-DPH-ceramide was rapidly anabolized mainly to sphingomyelin. In addition, small quantities of glucosylceramide were also formed. DPH-sphingosines were easily incorporated into plasma membranes of cultured fibroblasts and are likely to undergo flip flop since intracellular membranes also became labeled, when endocytosis was blocked at low temperature (7 degrees C). The N-hexanoyl-DPH-trans-sphingosine, C6-DPH-ceramide, like NBD-C6-ceramide (Lipsky, N. G., R. E. Pagano: Science 228, 745-747 (1985)) labeled intracellular membranes at 7 degrees C and predominantly Golgi membranes at 37 degrees C. Like NBD-C6-ceramide (Pagano, R. E., M. A. Sepanski, O. C. Martin: J. Cell Biol. 109, 2067-2079 (1989)) the C6-DPH-ceramide also stained the Golgi complex in prefixed cells whereas DPH-trans- and DPH-cis-sphingosine did not, indicating that it is the ceramide structure rather than the fluorophore itself which is responsible for this staining. DPH-sphingosine opens a way for chemical synthesis of DPH-glycolipids and DPH-sphingomyelin which would well serve as donors in fluorescence energy transfer experiments to study possible sphingolipid clustering in biological membranes.
首次合成了带有二苯基 - 1,3,5 - 己三烯基（DPH）作为荧光团的荧光D - 赤藓型鞘氨醇。研究了两种异构体，即DPH - 4（E） - 和DPH - 4（Z） - 鞘氨醇[（2S，3R） - 2 - 氨基 - 6 - （对 - （18 - 苯基） - 13,15,17（E，E，E） - 己三烯基）苯基己 - 4（E/Z） - 烯 - 1,3 - 二醇]，以及DPH - 4（E） - 鞘氨醇的N - 己酰基衍生物（C6 - DPH - 神经酰胺）在培养的人皮肤成纤维细胞中的分布和代谢。两种DPH - 鞘氨醇（4 - 反式和顺式）在活细胞中未显著酰化生成神经酰胺，但在体外可被小鼠脑微粒体蛋白转化为神经酰胺，不过比天然D - 赤藓型鞘氨醇慢。DPH - 4（Z） - 鞘氨醇的Km值与D - 赤藓型鞘氨醇相同（155μM），但Vmax值较低，为0.85而非1.9 nmol/mgh。更差的底物是DPH - 4（E） - 鞘氨醇，其Km为220μM，Vmax为0.81 nmol/mgh。在培养的人成纤维细胞中，C6 - DPH - 神经酰胺主要迅速合成代谢为鞘磷脂。此外，还形成了少量的葡糖神经酰胺。DPH - 鞘氨醇很容易掺入培养的成纤维细胞的质膜中，并且可能会发生翻转，因为当在低温（7℃）下阻断内吞作用时，细胞内膜也会被标记。N - 己酰基 - DPH - 反式鞘氨醇，C6 - DPH - 神经酰胺，与NBD - C6 - 神经酰胺（Lipsky，N.G.，R.E.Pagano：Science 228，745 - 747（1985））一样，在7℃时标记细胞内膜，在37℃时主要标记高尔基体膜。与NBD - C6 - 神经酰胺（Pagano，R.E.，M.A.Sepanski，O.C.Martin：J.Cell Biol.109，2067 - 2079（1989））一样，C6 - DPH - 神经酰胺也能对固定细胞中的高尔基体复合体进行染色，而DPH - 反式和顺式鞘氨醇则不能，这表明是神经酰胺结构而非荧光团本身导致了这种染色。DPH - 鞘氨醇为DPH - 糖脂和DPH - 鞘磷脂的化学合成开辟了一条途径，它们可作为荧光能量转移实验中的供体，用于研究生物膜中可能的鞘脂聚集。
