Comparison of the temperature sensitivity of protein synthesis by cell-free systems from liver of rat and skate (Raja ocellata)

Abstract

Studies were undertaken to determine the component(s) responsible for the temperature optimum characteristic of the protein-synthesizing system from skate and rat. 1. The macromolecular constituents of rat and skate liver ribosomes are compared. The number of ribosomal proteins is similar in the two species, although most proteins display different electrophoretic mobilities on polyacrylamide gels. The RNAs from the small subunit of skate and rat have similar sedimentation coefficients; however, the RNA from the large subunit of skate ribosomes appeared to be slight smaller than the comparable RNA from the rat. 2. Ribosomes from either rat or skate were capable of supporting poly(U)dependent polyphenylalanine synthesis with soluble factors from either species. 3. Maximal leucine incorporation directed by endogenous mRNA occurred at 35--40 degrees C with post-mitochondrial supernatant from the rat liver and at 20--30 degrees C with that from skate liver. 4. The characteristic temperature sensitivity of protein synthesis was dependent upon the source of cell sap and independent of the source of ribosomes. 5. Elongation factor 1 from both the rat and skate exhibited maximum activity at approx. 30 degrees C. 6. Phenylalanyl-tRNA synthetase from skate liver showed maximum activity at 30 degrees C while that from rat was maximally active at 37 degrees C. The rat enzyme, however, was active at 0--10 degrees C, at which temperature protein synthesis in the reconstructed rat system is virtually absent. 7. The protein-synthesizing capacity of the reconstituted system at various temperatures was closely correlated with the activity of Elongation factor 2 (translocase). Elongation factor 2 from rat liver displayed an optimum at 30 degrees C and lost all activity below 10 degrees C, while this same factor from skate liver showed an optimum at 20 degrees C and significant activity below 10 degrees C. At this low temperature the reconstituted skate liver system continued to exhibit the ability to synthesize protein. These studies suggest that Elongation factor 2 is the component responsible for determining the temperature at which the protein-synthesizing system displays its characteristic maximum activity.