We have utilized the binding of radioactive parathyroid hormone messenger ribonucleic acid (mRNA) to dog pancreatic microsomal membranes as an assay for the formation of membrane-bound ribosomes in vitro. Parathyroid hormone mRNA was labeled by RNA ligase catalyzed addition of cytidine 3',5'-[5'-32P]diphosphate to the 3' terminus. Radioactive parathyroid hormone mRNA was incubated in a reticulocyte cell-free protein-synthesizing system that was supplemented with dog pancreatic microsomal membranes. As determined by discontinuous sucrose gradient centrifugation, 25% of the total mRNA bound to the membranes in the reaction. Inhibition of protein synthesis by puromycin, addition of membranes after termination of the cell-free reaction, or disruption of the membranes after the reaction by deoxycholate resulted in less than 10% of the mRNA in the membrane region of the gradient. The binding of the mRNA to the membranes was resistant to incubation in 290 mM KCl, and only 25% of the mRNA was released by incubation in 540 mM KCl. This suggests that the nascent polypeptide chain is in part stabilizing the mRNA-ribosome membrane complex; this conclusion is strengthened by the observation that limited proteolysis caused the release of mRNA from the complex in high ionic strength buffer but not in low ionic strength buffer. Preformed mRNA-membrane complexes were disrupted within 5 min by addition of puromycin to the reaction, suggesting that direct interaction between the mRNA and membrane is not required to stabilize functional membrane-bound ribosomes. These experiments demonstrate that the binding of radioactive mRNA to microsomal membranes in vitro is a useful assay for analyzing the formation and nature of membrane-bound polysomes.
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