Ibrahim N G, Beattie D S
J Biol Chem. 1976 Jan 10;251(1):108-15.
Polysomes consisting of two to eight monosomes were isolated from yeast mitochondria by lysing the mitochondria with Triton X-100 and centrifugation in a 20 to 40% linear sucrose gradient. When yeast spheroplasts were pulse-labeled with [3H]-Leucine in the presence of cycloheximide to block cytoplasmic protein synthesis, radioactivity which was trichloroacetic acid-precipitable was present mainly in the polysome region. Incorporation of leucine was blocked by erythromycin, a specific inhibitor of mitochondrial protein synthesis. Release of radioactivity to the top of the gradient resulted from treating labeled polysomes with either puromycin or ribonuclease (in the latter case with the breakdown of polysomes), indicating that the radioactivity was present in nascent polypeptide chains. Yeast cells were grown in chloramphenicol for 3 hours and in fresh medium for 1 hour and then pulse-labeled with either [3H]leucine or [14C]formate. Three parameters showed a 2-fold increase in cells grown in chloramphenicol prior to pulse labeling: the polysome to monosome ratio, the amount of labeled precursor incorporated into proteins, and the rate of polypeptide chain initiation as judged by the formation of fMet-puromycin. Conversely, these parameters were all decreased approximately 50% in cells treated with cycloheximide prior to pulse labeling. Mitochondria were also isolated from cells previously grown in chloramphenicol or cycloheximide and incubated in vitro with [3H]leucine under optimal conditions. Acid-precipitable radioactivity in the polysome region was increased 3-fold in mitochondria from cells grown previously in chloramphenicol and decreased 75% in those grown in cycloheximide. Furthermore, chain initiation was deomonstrated in the isolated mitochondria by formation of fMet-puromycin. The rate of chain initiation in vitro was increased 2-fold in mitochondria isolated from chloramphenicol-treated cells.
通过用Triton X - 100裂解线粒体并在20%至40%的线性蔗糖梯度中离心，从酵母线粒体中分离出由两到八个单核糖体组成的多核糖体。当酵母原生质体在环己酰亚胺存在下用[³H] - 亮氨酸进行脉冲标记以阻断细胞质蛋白质合成时，可被三氯乙酸沉淀的放射性主要存在于多核糖体区域。亮氨酸的掺入被红霉素阻断，红霉素是线粒体蛋白质合成的特异性抑制剂。用嘌呤霉素或核糖核酸酶处理标记的多核糖体（在后一种情况下多核糖体会分解）导致放射性释放到梯度顶部，这表明放射性存在于新生多肽链中。酵母细胞在氯霉素中培养3小时，然后在新鲜培养基中培养1小时，接着用[³H]亮氨酸或[¹⁴C]甲酸进行脉冲标记。在脉冲标记前在氯霉素中培养的细胞中，三个参数增加了两倍：多核糖体与单核糖体的比例、掺入蛋白质中的标记前体的量以及通过甲酰甲硫氨酸 - 嘌呤霉素的形成判断的多肽链起始速率。相反，在脉冲标记前用环己酰亚胺处理的细胞中，这些参数均下降了约50%。线粒体也从先前在氯霉素或环己酰亚胺中培养的细胞中分离出来，并在最佳条件下与[³H]亮氨酸进行体外孵育。在先前在氯霉素中培养的细胞的线粒体中，多核糖体区域中酸可沉淀的放射性增加了3倍，而在环己酰亚胺中培养的细胞的线粒体中则下降了75%。此外，通过甲酰甲硫氨酸 - 嘌呤霉素 的形成在分离的线粒体中证明了链起始。从氯霉素处理的细胞中分离的线粒体中，体外链起始速率增加了2倍。
