Preparation of ribosome-free membranes from rat liver microsomes by means of lithium chloride.
1. Treatment of washed rat liver microsomes in a medium containing 0.12m-sucrose, 12.5mm-potassium chloride, 2.5mm-magnesium chloride and 25mm-tris-hydrochloric acid buffer, pH7.6, with 2m-lithium chloride at 5 degrees for 16hr. leads to the formation of membranes free of ribosomes and ribosomal subunits. 2. Confirmation of the absence of ribosomes from lithium chloride-prepared membranes was obtained by treatment of the membranes with sodium deoxycholate, followed by sucrose-density-gradient centrifugation, which showed the complete absence of ribosomes. 3. Treatment of membranes with phenol, followed by sucrose-density-gradient analysis of the isolated RNA, showed the presence of a small amount of 4s material. Repetition of the phenol extraction procedure in the presence of liver cell sap as a ribonuclease inhibitor again showed the presence of only 4s material. The 4s RNA was shown to be transfer RNA by the fact that it had the same capacity for accepting (14)C-labelled amino acids as isolated transfer RNA from rat liver pH5 enzyme. 4. Analysis showed that microsomes and membranes possessed similar glucose 6-phosphatase, NADH-2,6-dichlorophenol-indophenol reductase, NADH-neo-tetrazolium reductase, NADH-cytochrome c reductase and ribonuclease activities. 5. (3)H-labelled ribosomal RNA binds to membranes. However, isolation of the bound RNA by the phenol extraction procedure, followed by sucrose-density-gradient analysis, shows the RNA to be degraded to 7s material. Very little breakdown of (3)H-labelled ribosomal RNA bound to membranes occurs if the binding and isolation are carried out in the presence of liver cell sap.
D000818:Animals; D002247:Carbon Isotopes; D002499:Centrifugation, Density Gradient; D002712:Chlorides; D005952:Glucose-6-Phosphatase / Q000032:analysis; D008094:Lithium; D008566:Membranes; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000201:enzymology; D010088:Oxidoreductases / Q000032:analysis; D011485:Protein Binding; D012343:RNA, Transfer / Q000032:analysis; D051381:Rats; D012260:Ribonucleases / Q000032:analysis; D014316:Tritium
D002247:Carbon Isotopes; D002712:Chlorides; D014316:Tritium; D012343:RNA, Transfer; D008094:Lithium; D010088:Oxidoreductases; D012260:Ribonucleases; D005952:Glucose-6-Phosphatase