Borén J, Rustaeus S, Olofsson S O
Department of Medical Biochemistry, University of Göteborg, Sweden.
J Biol Chem. 1994 Oct 14;269(41):25879-88.
The mechanisms by which apolipoprotein B-100 (apoB-100) and apoB-48 assemble lipoproteins have been studied in the McA-RH7777 cell line. After incubation for 2 h with 360 microM oleic acid, the McA-RH7777 cells secreted apoB-100 mainly on very low density lipoprotein (VLDL) particles, as judged from sucrose gradient and sequential ultracentrifugation. ApoB-48 was secreted on both VLDL and on denser, high density lipoprotein (HDL)-like lipoproteins. Both apoB-48 and apoB-100 occurred on VLDL particles in the luminal content of the total microsomal fraction. In addition, both proteins were present on denser particles, in particular, those that banded in the HDL region. The denser particles containing apoB-48 were secreted from the cells, whereas those containing apoB-100 were retained in the cell and degraded. Pulse-chase experiments showed that apoB-100 on VLDL was present in the secretory pathway already after a labeling period as short as 3 min. Thus, this particle was the first apoB-100-containing lipoprotein that could be detected in the microsomal fraction of the cell. The assembly of labeled apoB-100 VLDL was acutely (within min) and completely blocked by cycloheximide, if the cycloheximide was added after the pulse labeling period. If, on the other hand, a 15-min chase was introduced after the labeling period, there was no effect of cycloheximide on the assembly of apoB-100 VLDL. A 15-min chase is enough to allow the pulse-labeled apoB nascent polypeptides to be completed to apoB-100. These results indicated that the assembly of apoB-100-containing VLDL is dependent upon ongoing protein biosynthesis during the time when the nascent polypeptide elongate. After this period, the assembly and secretion of the particles are independent of ongoing protein biosynthesis. The first apoB-48-containing particle seen in the luminal content of the microsomal fraction was the HDL-like particle. Pulse-chase experiments as well as experiments with different lengths of the radioactive pulse indicated that the formation of apoB-48-containing VLDL was delayed compared with the formation of the apoB-48-containing HDL-like particle and also in relation to the assembly of the different apoB-100-containing particles, including VLDL. After a 30-min pulse with [35S]methionine followed by a 120-min chase the secretory pathway of the cell was depleted of almost all lipoproteins except the HDL-like apoB-48-containing particle.(ABSTRACT TRUNCATED AT 400 WORDS)
在McA-RH7777细胞系中研究了载脂蛋白B-100（apoB-100）和apoB-48组装脂蛋白的机制。用360微摩尔油酸孵育2小时后，通过蔗糖梯度和连续超速离心判断，McA-RH7777细胞主要在极低密度脂蛋白（VLDL）颗粒上分泌apoB-100。apoB-48在VLDL以及密度更高的、类似高密度脂蛋白（HDL）的脂蛋白上分泌。在总微粒体部分的腔内容物中，apoB-48和apoB-100都存在于VLDL颗粒上。此外，两种蛋白质都存在于密度更高的颗粒上，特别是那些在HDL区域形成条带的颗粒。含有apoB-48的密度更高的颗粒从细胞中分泌出来，而含有apoB-100的颗粒则保留在细胞中并被降解。脉冲追踪实验表明，在短至3分钟的标记期后，VLDL上的apoB-100已经存在于分泌途径中。因此，这种颗粒是在细胞微粒体部分中可检测到的第一个含apoB-100的脂蛋白。如果在脉冲标记期后加入环己酰亚胺，标记的apoB-100 VLDL的组装会在数分钟内被急性且完全阻断。另一方面，如果在标记期后引入15分钟的追踪期，环己酰亚胺对apoB-100 VLDL的组装没有影响。15分钟的追踪期足以使脉冲标记的apoB新生多肽完成形成apoB-100。这些结果表明，含apoB-100的VLDL的组装在新生多肽延长期间依赖于正在进行的蛋白质生物合成。在此期间之后，颗粒的组装和分泌与正在进行的蛋白质生物合成无关。在微粒体部分的腔内容物中看到的第一个含apoB-48的颗粒是类似HDL的颗粒。脉冲追踪实验以及不同长度放射性脉冲的实验表明，含apoB-48的VLDL的形成与含apoB-48的类似HDL颗粒的形成相比有所延迟，并且与包括VLDL在内的不同含apoB-100颗粒的组装相比也有所延迟。在用[35S]甲硫氨酸进行30分钟脉冲后接着进行120分钟追踪，细胞的分泌途径中几乎所有脂蛋白都被耗尽，除了含apoB-48的类似HDL颗粒。（摘要截断于400字）
