On the cell-free system of protein synthesis from mammalian mitochondria

Abstract

Some properties of a submitochondrial cell-free system for protein synthesis are described. The system was prepared from rat liver mitochondria lysed with Triton X-100, and the lysate was characterized by a linear rate of [14C]amino acid incorporation for 15-20 min with subsequent decline in activity. The incorporation reaction was inhibited by chloramphenicol and was insensitive to cycloheximide. Poly(U) addition stimulated [14CA1phenylalanine incorporation by the preincubated submitochondrial system. Upon the addition of 7.5S mRNA that was isolated from mitochondria the major translation product was identified as a hydrophobic poly-peptide which in some properties (solubility in chloroform-methanol mixture) was similar to one of polypeptides synthesized by the sub-mitochondrial system on endogeneous mRNAs.

Isolation and partial characterization of poly (A)-containing 7.5S messenger RNA from rat liver mitochondria

“…Isolation of mitochondria. 1iitochondria were isolated as described (10) and treated with digitonin to remove cytoplasmic contaminations (11).…”

“…Poly(U)-Sbpharose chromatography was performed essentially as described earlier (11). RNA was dissolved in 20 mM tris-HCl buffer, pH 7.5 to adjust a final RNA concentration to 250-500 pg/ml.…”

“…Cell-free translation in a mitochondrial lysate RNA preparations were translated in a mitochondrial lysate from rat liver preincubated for 30 min to deplete the endogenous mlNA activity. The preparation of lysate and conditions for incubation were described earlier (11). The incubation was stopped with the addition of 80% methanol.…”

“…The electrophoresis of polypeptides synthesized in a cell--free system was performed in 7.5% polyacrylamide gels containing sodium dodecyl sulphate using tris-glycine buffer (11).…”

Enzymatic synthesis of DNA complementary to mitochondrial mRNA via reverse transcription

“…the reviews 1-3) and therefore, there is no restriction for synthesis of complemen- The mitochondrial mElA8 of rat liver were obtained as described earlier 10 from intramitochondrial polyribosomes with a subsequent isolation of the poly(A) containing fraction on the poly(U)-Sepharose. From the total mt-aRA a fraction was isolated soluble in 1.5 N NaCl.…”