Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions.
Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions.
复制标题
DOI：
--
复制
发表时间：
1991-03
影响因子：
6.5
通讯作者：
Robert L. Hamilton;A. Moorehouse;Richard J. Havel
中科院分区：
文献类型：
--
作者：
Robert L. Hamilton;A. Moorehouse;Richard J. Havel
使用两种方法从大鼠肝脏中分离肝细胞高尔基体部分。一个程序产生轻质高尔基体部分（GF1 + 2）和其他“完整”的水池堆。通过静脉注射[3H]棕榈酸酯来标记新生极低密度脂蛋白（VLDL）中的甘油三酯脂肪酸，并注射放射性标记的脂蛋白作为潜在污染内体的标记物。 GF1 + 2 级分的内体标记物富集了许多倍，表明存在大量的内体污染，而来自同一肝脏的完整高尔基体级分大约有 7% 被污染。通过电子显微镜观察，GF1 + 2 级分主要含有多囊泡体 (MVB)，以及一些高尔基体衍生的分泌囊泡。通过对程序的简单修改，去除了大部分夹带的内体，进一步减少了完整高尔基体部分的小内体污染。从这些高度纯化的完整高尔基体级分中释放的高尔基体 VLDL（d 小于 1.010 g/ml）的表面成分与血浆 VLDL 的表面成分不同。高尔基体 VLDL 中的未酯化胆固醇含量是血浆 VLDL 的五倍，但磷脂含量却是血浆 VLDL 的两倍。高尔基体 VLDL 和血浆 VLDL 含有相似含量的胆固醇酯和甘油三酯。高尔基体VLDL的蛋白质含量明显低于血浆VLDL。 ApoB-100 和 apoB-48 的表现相似，但新生 VLDL 含有较少的 C 载脂蛋白。 ApoA-I 主要作为高尔基体 VLDL 中的前蛋白存在，但在血浆 VLDL 中几乎缺乏。 ApoE 约占高尔基体 VLDL 以及血浆 VLDL 蛋白质质量的 22%； apoE亚型的分布也相似。从破裂的高尔基体池释放的载脂蛋白E和pro A-I大部分与高尔基体VLDL结合或与高尔基体膜相关。通过电子显微镜在完整高尔基体级分的内容物中没有看到类似低密度脂蛋白（LDL）和高密度脂蛋白（HDL）的颗粒。这些观察结果表明，新生高尔基体VLDL是肝细胞来源的大鼠血浆脂蛋白的主要颗粒前体，并表明具有血浆HDL和LDL密度的颗粒不存在于正常肝细胞的分泌途径中。因此，这项关于未受污染的肝细胞高尔基体部分中所含新生血浆脂蛋白前体特性的研究结果与之前发表的工作有很大不同。
Two procedures were used to isolate hepatocytic Golgi fractions from rat liver. One procedure yields a light Golgi fraction (GF1 + 2) and the other "intact" stacks of cisternae. Triglyceride fatty acids in nascent very low density lipoproteins (VLDL) were labeled by injection of [3H]palmitate intravenously, and radiolabeled lipoproteins were injected as markers of potentially contaminating endosomes. GF1 + 2 fractions were enriched manyfold in the endosomal markers, indicative of substantial endosomal contamination, whereas intact Golgi fractions from the same livers were about 7% as contaminated. By electron microscopy, GF1 + 2 fractions contained mainly multivesicular bodies (MVBs), together with some Golgi-derived secretory vesicles. The small endosomal contamination of intact Golgi fractions was further reduced by a simple modification of the procedure, which removed most entrained endosomes. The surface constituents of Golgi VLDL (d less than 1.010 g/ml) released from these highly purified intact Golgi fractions differed from those of plasma VLDL. Golgi VLDL contained fivefold less unesterified cholesterol than plasma VLDL, but twofold more phospholipids. Golgi VLDL and plasma VLDL contained similar amounts of cholesteryl esters and triglycerides. The protein content of Golgi VLDL was substantially lower than that of plasma VLDL. ApoB-100 and apoB-48 were similarly represented, but nascent VLDL contained less of the C apolipoproteins. ApoA-I was present mainly as the proprotein in Golgi VLDL, but was virtually lacking in plasma VLDL. ApoE comprised about 22% of the protein mass of Golgi VLDL as well as plasma VLDL; the distribution of apoE isoforms was also similar. Apolipoproteins E and pro A-I released from ruptured Golgi cisternae were largely bound to the Golgi VLDL or were associated with Golgi membranes. Particles resembling low density lipoproteins (LDL) and high density lipoproteins (HDL) were not seen by electron microscopy in contents of intact Golgi fractions. These observations indicate that nascent Golgi VLDL are the primary particulate precursors of rat plasma lipoproteins of hepatocytic origin, and suggest that particles with the density of plasma HDL and LDL do not exist within the secretory pathway of normal hepatocytes. Thus, the results of this research on the properties of nascent plasma lipoprotein precursors contained within uncontaminated hepatocytic Golgi fractions differ substantially from previous published work.