Adeli K, Theriault A
Department of Chemistry and Biochemistry, University of Windsor, Ont., Canada.
Biochem Cell Biol. 1992 Dec;70(12):1301-12. doi: 10.1139/o92-177.
Insulin modulation of apolipoprotein B gene expression was studied at the translational level by the use of a cell-free translation system from a hepatoma cell-line, HepG2. Extracts of HepG2 cells lysed with lysolecithin were found to have high in vitro protein synthesizing activity utilizing endogenous mRNA. The level of peptide chain initiation was high, as suggested by a significant inhibition of translation by edeine. The translation products of endogenous mRNA in HepG2 cell-free lysate were probed with anti-apolipoprotein B antibodies to investigate its synthesis. A 550 kilodalton (kDa) polypeptide was selected by a polyclonal antibody, as well as a monoclonal antibody, against the C-terminal end of apolipoprotein B molecule. This in vitro synthesized polypeptide was also found to compare well in size with the in vivo product. The HepG2 lysate was also shown to efficiently synthesize in vitro a number of other proteins including albumin, apolipoprotein E, apolipoprotein A1, and actin. The in vitro synthesis of polypeptides as large as 500 kDa was unexpected and has not previously been demonstrated in a cell-free system. The HepG2 translation system was used to investigate the effect of insulin on the in vitro translation of apolipoprotein B. Lysates prepared from HepG2 cells treated with insulin were found to have lower translational activity (by an average of 52.3%) for apolipoprotein B compared with lysates from control untreated cells. In vitro synthesis of actin and apolipoprotein E were unaffected under these conditions. The insulin-stimulated decline in in vitro apolipoprotein B synthesis was not due to a change in apolipoprotein B mRNA levels as determined by slot- and Northern-blot analyses, suggesting that the inhibitory effect of insulin may be exerted partly at the level of apolipoprotein B mRNA translation.
利用肝癌细胞系HepG2的无细胞翻译系统，在翻译水平上研究了胰岛素对载脂蛋白B基因表达的调节作用。发现用溶血卵磷脂裂解的HepG2细胞提取物利用内源性mRNA具有较高的体外蛋白质合成活性。如依地碱对翻译有显著抑制作用所表明的，肽链起始水平较高。用抗载脂蛋白B抗体探测HepG2无细胞裂解物中内源性mRNA的翻译产物，以研究其合成情况。一种针对载脂蛋白B分子C末端的多克隆抗体以及单克隆抗体选择出了一种550千道尔顿（kDa）的多肽。还发现这种体外合成的多肽在大小上与体内产物相当。HepG2裂解物还被证明能在体外高效合成许多其他蛋白质，包括白蛋白、载脂蛋白E、载脂蛋白A1和肌动蛋白。能在体外合成高达500 kDa的多肽出乎意料，此前在无细胞系统中尚未得到证实。HepG2翻译系统用于研究胰岛素对载脂蛋白B体外翻译的影响。发现用胰岛素处理的HepG2细胞制备的裂解物与未处理的对照细胞裂解物相比，载脂蛋白B的翻译活性较低（平均降低52.3%）。在这些条件下，肌动蛋白和载脂蛋白E的体外合成未受影响。胰岛素刺激下载脂蛋白B体外合成的下降并非由于通过狭缝印迹和Northern印迹分析确定的载脂蛋白B mRNA水平的变化，这表明胰岛素的抑制作用可能部分在载脂蛋白B mRNA翻译水平上发挥作用。
