A simple and rapid procedure for high-yield isolation of essentially undegraded free and membrane-bound polysomes from rat liver.
A procedure is described for the preparation of free and membrane-bound polysomes from rat liver. The procedure involves: differential centrifugation of liver homogenate to separate free and membrane-bound polysomes; treatment of the membrane-bound polysome fraction with a detergent to release bound polysomes from membranes; and magnesium precipitation of both classes of polysomes. Free and bound polysomes prepared in this manner were essentially undegraded and highly active in cell-free protein synthesis. The recovery of polysomes was nearly quantitative and the distribution between the free and membrane-bound state was 41 and 59%, respectively. Polypeptides synthesized in vitro by the free and membrane-bound polysomes were quite different. The majority (81-84%) of mRNA activities of two secretory proteins (albumin and transferrin) were recovered in the membrane-bound polysomes, whereas the majority (81-85%) of mRNA activities of two cytosolic [aldolase B, EC 4.1.2.13, and argininosuccinate synthetase, EC 6.3.4.5], one mitochondrial [ornithine carbamoyltransferase, EC 2.1.3.3] and one peroxisomal [catalase, EC 1.11.1.6] proteins were recovered in the free polysomes. A polysome class synthesizing ornithine carbamoyltransferase was purified 42-fold from the free polysomes by immunoprecipitation. The procedure is rapid (4-5 h) and reproducible, and provides a nearly quantitative means of separating the two classes of polysomes.
D000818:Animals; D002451:Cell Compartmentation; D002458:Cell Fractionation / Q000379:methods; D011232:Chemical Precipitation; D003593:Cytoplasm / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000648:ultrastructure; D008274:Magnesium; D008297:Male; D011132:Polyribosomes; D011189:Potassium Chloride / Q000494:pharmacology; D014176:Protein Biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains; D014461:Ultracentrifugation
D011189:Potassium Chloride; D008274:Magnesium