Isolation of release factor from mammalian liver and the use of rat liver ribosomes in the release assay

Abstract

1. Release factor assayed by the method of Caskey was isolated and purified from the supernatant fraction of rabbit liver homogenates. A molecular weight of approximately 350 000 was determined for the release factor. The significance of this finding is discussed in relation to previously reported determinations. 2. Ribosomes obtained from the non-membrane bound fraction ( i.e. free ribosomes) of rat liver can be used as a substitute for reticulocyte ribosomes in the preparation of the f[ 3H]Met-tRNA fMet ( Escherichia coli) · ribosome intermediate which is used to measure fMet release. A comparison of preparations of reticulocyte ribosome intermediates and rat liver free ribosome intermediates revealed that in both preparations the release of fMet depended upon the presence of release factor, poly(U, A 3) and GTP. 3. The binding of fMet-tRNA fMet to rat liver ribosomes is highly specific even at high magnesium levels. Formylation of Met-tRNA fMet is shown to be essential for puromycin reactivity of the Met-tRNA fMet rat liver ribosome intermediate. In addition, the results show that rat liver free ribosomes are more efficient in this respect than deoxycholate-prepared ribosomes.