Dissous C, Lempereur C, Verwaerde C, Krembel J
Eur J Biochem. 1978 Feb 1;83(1):17-27. doi: 10.1111/j.1432-1033.1978.tb12063.x.
Techniques allowing the recovery of large free and membrane-bound polysomes in high yield are reported. Subcellular fractions were prepared from rat liver homogenates as described in the preceding paper. Purified microsomal membranes (obtained from the post-lysosomal supernatant) were adjusted to 50 mM Mg(CH3COO)2 and treated with 2% Triton X-100 and 0.3% sodium deoxycholate in the presence of yeast RNA and cell sap, and polysomes were purified by overnight centrifugation through low-ionic-strength discontinuous sucrose gradients containing 2 mg/ml of cell sap proteins. Polysomes were isolated from the mitochondria/endoplasmic reticulum complex (fraction C) by treatment with 2% Triton X-100 and 0.5% sodium deoxycholate in the presence of 50 mM Tris-HCl, pH 7.6, 0.1 M KCl, 0.15 M NH4Cl and 50 mM Mg(CH3COO)2 and purified through sucrose layers of decreasing ionic strength containing 2 mg/ml of cell sap proteins. Analyses of polysomes in isokinetic sucrose gradients showed that the free polysome fraction and both membrane-bound polysome fractions had 14-15 ribosomes per mRNA at the maximum of absorbance. Experiments from which these methods were derived are described.
报道了能够高产回收大量游离和膜结合多核糖体的技术。如前文所述，从大鼠肝脏匀浆中制备亚细胞组分。将纯化的微粒体膜（从溶酶体后上清液中获得）调整至50 mM Mg(CH3COO)2，并在酵母RNA和细胞液存在的情况下用2% Triton X-100和0.3%脱氧胆酸钠处理，然后通过含有2 mg/ml细胞液蛋白的低离子强度不连续蔗糖梯度过夜离心来纯化多核糖体。通过在50 mM Tris-HCl（pH 7.6）、0.1 M KCl、0.15 M NH4Cl和50 mM Mg(CH3COO)2存在的情况下用2% Triton X-100和0.5%脱氧胆酸钠处理，从线粒体/内质网复合物（组分C）中分离多核糖体，并通过含有2 mg/ml细胞液蛋白的离子强度递减的蔗糖层进行纯化。在等速蔗糖梯度中对多核糖体的分析表明，在吸光度最大值时，游离多核糖体组分以及两个膜结合多核糖体组分在每个mRNA上均有14 - 15个核糖体。描述了衍生出这些方法的实验。
