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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.
Insights
New techniques efficiently recover large free and membrane-bound polysomes from rat liver. These methods yield high quantities of polysomes, crucial for studying protein synthesis and cellular function.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Polysomes, the complexes of messenger RNA (mRNA) and ribosomes, are central to protein synthesis.
- Efficient isolation of both free and membrane-bound polysomes is essential for comprehensive analysis of cellular translation.
Purpose of the Study:
- To develop and report high-yield techniques for the recovery of large free and membrane-bound polysomes.
- To enable detailed investigation into the composition and function of polysomal complexes.
Main Methods:
- Subcellular fractionation of rat liver homogenates.
- Treatment of microsomal membranes and endoplasmic reticulum complexes with detergents (Triton X-100, sodium deoxycholate) and salts.
- Purification of polysomes using discontinuous sucrose gradient centrifugation in low-ionic-strength conditions with cell sap proteins.
Main Results:
- Successful isolation of both free and membrane-bound polysomes in high yield.
- Analysis revealed approximately 14-15 ribosomes per mRNA in purified polysome fractions.
- The described methods are effective for separating and purifying distinct polysome populations.
Conclusions:
- The reported techniques provide a robust method for obtaining large free and membrane-bound polysomes.
- These methods facilitate further research into translational control and protein synthesis in eukaryotic cells.