Free and Membrane-Bound Polysomes from Rat Liver. 2. Recovery of Large Free and Membrane-Bound Polysomes

Abstract

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.