Jiang L W, Mitchell B A, Teodoro J G, Rip J W
Department of Biochemistry, University of Western Ontario, Canada.
Biochim Biophys Acta. 1993 Apr 22;1147(2):205-13. doi: 10.1016/0005-2736(93)90005-k.
We are using fluorescent derivatives to visualize the endocytic transport of dolichol intermediates from the cell surface to the lysosome, and to estimate their rate of turnover within the lysosome. Anthroyl dolichol and anthroyl [1-14C]dolichol were synthesized and purified by chromatography on silica and C18 Sep-Paks followed by high-performance liquid chromatography on C18. The successful synthesis of anthroyl polyisoprenoid alcohols was confirmed by the use of uv-visible spectrometry and by fluorescence spectrometry. The purified esters were taken up into Ham's media containing 10-30% fetal calf serum or alternatively reconstituted into phospholipid liposomes for delivery to human fibroblasts in culture. The uptake of fluorescent dolichol esters into the cells and into lysosomes was demonstrated using fluorescence microscopy. The localization of anthroyl dolichol in lysosomes was further documented by simultaneously labeling fibroblasts with anthroyl dolichol and FITC-dextran a recognized lysosomal marker. Fibroblasts generally showed several groupings (domains) of lysosomes, some were dually labeled while others were labeled exclusively with either anthroyl dolichol or FITC-dextran. Labeling with anthroyl dolichol was very slow relative to labeling of the same fibroblasts with FITC-dextran suggesting that anthroyl dolichol acts as a labeling agent for intracellular membranes, particularly those of the lysosome while the dextran fluorescence is presumably of lysosolic origin. Several types of experiments were done with anthroyl [1-14C]dolichol to establish that the fluorescence seen in lysosomes represents anthroyl dolichol. Anthroyl dolichol appears to enter fibroblasts intact, since we were unable to recover any free [1-14C]dolichol from total lipid extracts of (i) media used for the uptake of anthroyl dolichol or (ii) the media removed from cells labelled for 42 h. In addition, attempts to hydrolyze anthroyl [1-14C]dolichol in vitro using whole fibroblast homogenates at pH 4.0 and 7.5 were unsuccessful, even though the fibroblasts expressed acid lipase activity using 4-methylumbelliferyl palmitate as substrate.
我们正在使用荧光衍生物来观察多萜醇中间体从细胞表面到溶酶体的内吞运输，并估计它们在溶酶体内的周转速率。合成了蒽基多萜醇和蒽基[1-¹⁴C]多萜醇，并通过硅胶柱色谱和C18 Sep-Pak柱色谱进行纯化，随后在C18柱上进行高效液相色谱分析。通过紫外可见光谱法和荧光光谱法证实了蒽基多异戊二烯醇的成功合成。将纯化的酯加入含有10%-30%胎牛血清的Ham培养基中，或者重新组装成磷脂脂质体，用于递送至培养的人成纤维细胞。使用荧光显微镜观察到荧光多萜醇酯被细胞和溶酶体摄取。通过同时用蒽基多萜醇和FITC-葡聚糖（一种公认的溶酶体标记物）标记成纤维细胞，进一步证明了蒽基多萜醇在溶酶体中的定位。成纤维细胞通常显示出几组（区域）溶酶体，一些被双重标记，而另一些仅被蒽基多萜醇或FITC-葡聚糖单独标记。与用FITC-葡聚糖标记相同的成纤维细胞相比，用蒽基多萜醇标记非常缓慢，这表明蒽基多萜醇作为细胞内膜的标记剂，特别是溶酶体膜的标记剂，而葡聚糖荧光可能来自溶酶体。用蒽基[1-¹⁴C]多萜醇进行了几种类型的实验，以确定在溶酶体中看到的荧光代表蒽基多萜醇。蒽基多萜醇似乎完整地进入成纤维细胞，因为我们无法从（i）用于摄取蒽基多萜醇的培养基或（ii）从标记42小时的细胞中去除的培养基的总脂质提取物中回收任何游离的[1-¹⁴C]多萜醇。此外，即使成纤维细胞以4-甲基伞形酮棕榈酸酯为底物表达酸性脂肪酶活性，尝试在pH 4.0和7.5下使用全成纤维细胞匀浆在体外水解蒽基[1-¹⁴C]多萜醇也未成功。
