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Repeated low speed centrifugations of a post-mitochondrial supernatant from rat liver through discontinuous sucrose gradients in presence of cytoplasmic RNase inhibitor yield a purified rough endoplasmic reticulum fraction. This fraction contains loosely-associated ribosomes (up to 20%) which can be released from membranes by a slight RNase treatment.

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.

Substantial improvements of cellular fractionation in ionic conditions allowing preservation of polysome structure and polysome-membrane interactions are reported. They consist primarily in minimizing the lysosomal content of fractions containing endoplasmic reticulum by isolating a lysosome-rich fraction with little loss (6%) of RNA. Endoplasmic reticulum membranes were recovered in high yield, mainly in association with mitochondria, the remainder being found in the post-lysosomal supernatant. The latter also contained practically all the free polysomes, as judged by metrizamide gradient analyses. The distributions of various constituents (RNA, DNA, protein and marker enzymes) among cell fractions is presented.

The behaviour of rat liver cytoplasmic ribonucleoproteins in metrizamide has been studied to determine whether the iodinated compound would offer any advantage over other centrifugation media for studies of polysome structure and function. 1. Polysomes had a density of 1.295--1.300 g/cm3 in metrizamide gradients which was also the density of glycogen. However, the polysaccharide reached its equilibrium more rapidly than the polysomes. Thus a short centrifugation of a polysome suspension from non-starved rats over a 40% metrizamide cushion was sufficient to eliminate more than 85% of the glycogen with a polysome yield of about 75%. 2. Ribosomal subunits had neighbouring densities (1.23 and 1.20 g/cm3 for large and small EDTA-derived subunits; 1.23 and 1.21 g/cm3 for large and small KCl/puromycin-derived subunits, respectively). Polysomal messenger ribonucleoproteins were heterogeneously distributed (phi = 1.12 to 1.23 g/cm3) and overlapped with subunits in a similar manner as in sucrose gradients. 3. Analysis of a post-mitochondrial supernatant in metrizamide showed a clear separation of free polysomes, rough membranes and the soluble phase.