1. 1. The effect of salt-free hydroxylamine (NH 2OH) on rabbit-reticulocyte ribosomes labeled by prior incubation with [ 14C]amino acids has been investigated and compared with the effect of puromycin (stylomycin) in order to study the mode of attachment of nascent polypeptides to the ribosome. 2. 2. Under proper conditions, NH 2OH is found to release labeled polypeptides from the ribosome without concomitant disintegration of the ribosome. 3. 3. Incubation of the labeled ribosome in a suitable cell-free system with completion and release of the labeled polypeptide into the soluble supernatant as hemoglobin produces a proportional decrease in the fraction available for release by NH 2OH. 4. 4. The fraction released by NH 2OH appears to coincide with the fraction released by puromycin as treatment with either agent renders the ribosomal label unsusceptible to further release by the other agent. Electrophoretic patterns of the polypeptides released by both agents are similar, but unlike hemoglobin. 5. 5. The concentration where maximal release occurs, 3 M NH 2OH and the time course, with maximal release at 8-10 min, resemble the reaction of NH 2OH with aminoacyl transfer RNA. 6. 6. It is concluded that NH 2OH, like puromycin, may release the polypeptide chain from the ribosome by breaking the ester bond linking the polypeptide at its C-terminal acyl group to the ribosome-bound transfer RNA.
Bibliographical noteFunding Information:
Support was provided by a grant from the National Cancer Institute, National Institutes of Health (Contract No. CRTY 5o18).