The isolation of large polysomes in high yield from unfractionated tissue homogenates.
It was found that if large quantities of both exogenous RNA and Mg-2+ were present during gentle tissue homogenization, the subsequent addition of deoxycholate to the whole homogenate produced a viscous mass from which polysomes could be isolated in large yields. These polysomes were substantially less degraded than those isolated by previous methods. In the case of rat liver, 15 ribosomes per mRNA was the species present in highest concentration. The parameters of this method were investigated and optimized. About 80 percent of the rRNA in the homogenates was recovered in the polysomes. Omission of deoxycholate permitted the isolation of less-degraded free polysomes as well. In the liver of fed rats these represented one-fourth of the total polysomes, in good agreement with results obtained by an independent approach. Using the method to isolate polysomes from the liver of starving rats, it was found that only about one percent of the large amount of monomers and dimers present resulted from polysome breakdown during isolation. It was further shown that random RNAase hydrolysis of polysomes could not produce the patterns of liver polysomes seen during starvation. Polysomes isolated by this procedure were quite stable in solution and were very active in cell-free protein synthesis. Application of this method without adaptation to eight other tissues also permitted the isolation of large polysomes in high yields.
D000818:Animals; D001921:Brain / Q000648:ultrastructure; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D003840:Deoxycholic Acid; D007668:Kidney / Q000648:ultrastructure; D008099:Liver / Q000648:ultrastructure; D008274:Magnesium; D008325:Mammary Neoplasms, Experimental; D051379:Mice; D009928:Organ Specificity; D010902:Pituitary Gland / Q000648:ultrastructure; D011132:Polyribosomes / Q000648:ultrastructure*; D012313:RNA; D051381:Rats; D012270:Ribosomes / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae; D013217:Starvation; D013950:Thymus Gland / Q000648:ultrastructure
D003840:Deoxycholic Acid; D012313:RNA; D008274:Magnesium