Open AccessFree and Membrane‐Bound Polysomes from Rat Liver
Author(s) -
DISSOUS Claude,
LEMPEREUR Colette,
VERWAERDE Claudie,
KREMBEL Jean
Publication year - 1978
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1978.tb12063.x
Subject(s) - polysome , microsome , centrifugation , membrane , endoplasmic reticulum , biochemistry , sucrose , ribosome , chromatography , cell fractionation , chemistry , biology , rna , in vitro , gene
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(CH 3 COO) 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 NH 4 Cl and 50 mM Mg(CH 3 COO) 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.