Dataset Information

Characterization of cell-free protein-synthesis systems from undeveloped and developing Artemia embryos.

ABSTRACT: We have developed and characterized translationally active cell-free systems from Artemia embryos at different developmental times. The optimized lysates from 16 h-developed embryos incorporated radiolabelled amino acids into polypeptides for up to 120 min. The polypeptides synthesized ranged in Mr from 150,000 to 10,000, suggesting that the endogenous mRNA was capable of directing the synthesis of complete polypeptides. Similar results were obtained by using lysates from early developmental stages; even the cell-free system prepared from 1 h-developed embryos was partially active in protein synthesis. Furthermore, all these lysates were capable of re-initiation, as demonstrated by inhibition of initiation with the inhibitors edeine and 7-methylguanosine 5'-triphosphate. Because we found no endogenous protein-synthetic activity in the corresponding lysates from undeveloped embryos, we have used cell-free translation systems from 0 h- and 16 h-developed Artemia embryos to analyse the mechanisms limiting protein synthesis at very early developmental stages. Undeveloped-embryo lysates supplemented with nuclease-treated reticulocyte lysate were capable of translating endogenous mRNAs to give products with a wide spectrum of Mr values, but lysates of 16 h-developed embryos supplemented in this way were not further stimulated. The nuclease-treated lysate appeared to be unnecessary 5 h after resumption of development. These results suggested that a component(s) limiting translation in the undeveloped-embryo lysate was provided by the nuclease-treated reticulocyte lysate, and that this component(s) no longer limited protein synthesis after development. In view of these results, partially fractionated reticulocyte lysates were tested for restoration of protein-synthetic activity in the undeveloped embryo lysate. A high-salt ribosomal wash devoid of ribosomal subunits, which is considered a crude polypeptide-initiation-factor preparation, also restored translation activity in the undeveloped embryo lysate and made it capable of directing the synthesis of both endogenous mRNAs and exogenous (globin) mRNA.

SUBMITTER: Moreno A

PROVIDER: S-EPMC1151076 | biostudies-other | 1991 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:To identify factors responsible for the down-regulation of mitochondrial biosynthetic processes during anoxia in encysted Artemia franciscana embryos, the effects of oxygen limitation and pH on protein synthesis were investigated in isolated mitochondria. At the optimal pH of 7.5, exposure of mitochondria to anoxia decreases the protein synthesis rate by 79%. Rates were suppressed by a further 10% at pH 6.8, the intracellular pH (pHi) measured under anoxia in vivo. Matrix pH, measured under identical conditions, was 8.43 +/- 0.01 at an extra-mitochondrial pH of 7.9 (mean +/- S.E.M., n = 3), 8.05 +/- 0.01 at pH 7.5, and 7.10 +/- 0.01 at pH 6.8. The matrix pH did not vary (P > or = 0.20) as a function of oxygen availability during the 1 h assays. Intramitochondrial purine nucleotides varied little as a function of pH. In contrast, after 1 h of protein synthesis under anoxia, ATP levels decreased by up to 40%, whereas AMP, ADP and GDP concentrations increased, and GTP and GMP concentrations remained relatively constant. The addition of 1 mM ATP at the onset of anoxia maintained the ATP/ADP ratio at the aerobic value, but did not stabilized the GTP/GDP ratio or rescue rates of protein synthesis. Thus, at present, we cannot eliminate the possibility that the decrease in the GTP/GDP ratio during anoxia may contribute to the suppression of protein synthesis. The effect of anoxia was reversible; the rate of protein synthesis upon reoxygenation after a 30 min bout of anoxia was comparable (P = 0.14) with the pre-anoxic rate (193 +/- 17 and 174 +/- 6 pmol of leucine per mg of protein respectively, mean +/- S.E.M., n = 3). The array of mitochondrial translation products did not differ qualitatively as a function of either oxygen availability or pH. Finally, similar pH profiles for protein synthesis were obtained with either [3H]leucine or [3H]histidine (known to use different transporters). Consequently, it is improbable that the pH-sensitivity of protein synthesis can be explained by a specific protein effect on the import of the radiolabelled amino acid used. In summary, both oxygen limitation and acidic pH suppress rates of mitochondrial protein synthesis and are likely to contribute to the arrest of mitochondrial anabolic processes during anoxia-induced quiescence in A. franciscana embryos.

Dataset Information

Characterization of cell-free protein-synthesis systems from undeveloped and developing Artemia embryos.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets