| Abstract: | Total rough microsomes, isolated from the dog pancreas, were stripped of membranes-bound polysomes by treatment with either EDTA or puromycin and 0.5 M KCl. The stripped microsomal membranes were isolated relatively free from contamination, by using buoyant density centrifugation, and mRNA was isolated from both the membrane fraction and the released material. Depending on the method used to strip the rough microsomes, we found a variable but small percentage (3--15%) of the cellular poly(A)-containing mRNA attached to the microsomal membranes. Reextraction of isolated microsomal membranes with puromycin and 0.5 M KCl reduced the content of membrane-associated mRNA by approximately 50%, resulting in less than 2% of the total membrane-bound polysomal mRNA remaining associated with the microsomal membranes. The membrane-associated mRNA was characterized by translation in the wheat germ cell-free protein synthesizing system, and the products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The translation products of the membrane-associated mRNA were identical with those from the total pancreas mRNA and also with those obtained by using mRNA isolated from material released directly from the rough microsomes. |
