Isolation and Characterization of Rat‐Liver Mitochondrial Ribosomes Highly Active in Poly(U)‐Directed Polyphenylalanine Synthesis

Abstract

The procedure used to obtain mitochondrial ribosomes from rat liver highly active in catalyzing a poly(U)-dependent polyphenylalanine synthesis is described.Two kinds of particles with sedimentation coefficients of 80-S and 55-5, present in the mitochondrial lysate, are able to catalyze protein synthesis in vitro.Sensitivity to specific inhibitors of protein synthesis demonstrates that the activity of 55-5 particles is only of niitochondrial origin whereas that of 80-5 particles is partly due to mitochondrial ribosomes and partly to contaminating cytoplasmic ribosomes.Characterization of the two kinds of mitochondrial particles has been carried out by studying their sedimentation pattern in different experimental conditions and by analyzing the RNA extracted by polyacrylamide gel electrophoresis. 16-S and 12-5 RNA seem to be specific mitochondrial ribosomal species.The results suggest that the 56-5 particle represents the basic unit for protein synthesis in rat liver mitochondria. Particles with higher sedimentation value probably represent aggregates of mitochondrial monosomes.

Mammalian Mitochondrial Ribosomes. Studies on the Exchangeability of Polypeptide Chain Elongation Factors from Bacterial and Mitochondrial Systems

“…The findings of other research groups who achieved activity in vitro with 55-S ribosomes and supernatant fraction from E. coli [15,16] are in apparent contradiction to our results. However, no evidence was presented in these investigations that the mitochondrial ribosomes treated with only 100 mM KCI were really free of endogenous elongation factors.…”

The restriction fragment map of rat-liver mitochondrial DNA: A reconsideration

“…55-S mitochondrial ribosomes were isolated as described elsewhere [5], the rRNAs were extracted from the ribosomes with a SDS/phenol procedure. Poly(A)-containing mtRNA was obtained by oligo(dT) cellulose chromatography, as presented in detail recently [6].…”

Purification and partial characterization of mitochondrial ribosomes from a HeLa cell line resistant to chloramphenicol

“…The exact molecular mechanism of this resistance is not known but it has been suggested that either some aspect of the secondary structure of the messenger (poly(U)) or the hydrophobicity or aromaticity of the amino acid (phenylalanine) allows this aminoacyl-tRNA to compete successfully with chloramphenicol for occupation of the A site on the ribosome (Pestka, 1977). Nonetheless, Ibrahim et al (1974) and Greco et al (1973) both reported that chloramphenicol inhibited poly(U)-directed polypeptide synthesis by mitochondrial ribosomes vitro. Avadhani and Rutman (1974) reported that in an S-25 prepared from Ehrlich acites cell mito chondria, synthesis of polyphenylalanine was inhibited by chloramphenicol.…”

“…The preparations were free of cytoplasmic and bacterial contamination but were not active in in vitro polypeptide synthesis. In 1973, Ibrahim et al, using a modification of the procedure of O'Brien and Kalf (1967), and Greco et al (1973), were able to isolate rat liver mitochondrial ribosomes which were active in poly(U)-directed polyphenylalanine synthesis and which were more active than the preparation of O'Brien and Kalf in tiie peptidyl transferase assay. Both systems used ^ coli supernatant factors and were inhibited by low levels of chloramphenicol.…”

“…Both systems used ^ coli supernatant factors and were inhibited by low levels of chloramphenicol. Greco et al (1973) purified their mitochondria using three washes in 1 mM EDTA rather than the extensive washing through discontinuous gradients used by Ibrahim et al (1973), but both preparations still required sucrose gradient purification to remove cytoplasmic ribosomes. In that same year, DeVries and Van der Koogh-Schuuring (1973) (Ibrahim et , 1973),…”