A critical study of amino acid incorporation into protein by isolated liver mitochondria from adult rats.
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ABSTRACT:
SUBMITTER: Sandell S
PROVIDER: S-EPMC1270623 | biostudies-other | 1967 Aug
REPOSITORIES: biostudies-other
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Project description:1. The incorporation of amino acids into protein in isolated mitochondria has now been studied in more detail, and with mitochondria from a range of tissues. 2. Liver mitochondria from newborn rats are twice as active as those from adult rats. 3. The mitochondria are inactivated by excessive homogenization and repeated freezing and thawing. 4. The incorporation is sensitive to conditions of incubation and in particular to the rate of oxygenation, shape of vessel and depth of fluid. Best results are obtained by incubation in flat-bottomed vessels containing suspensions with less than 3mm. depth of fluid. 5. The requirement for oxidizable substrates has been examined with a range of substances, and most of the common energy-yielding metabolites of the mitochondrion are effective. Their activity is greatly influenced by concentration, and some, but not all, of the substrates show optimum concentrations for incorporation with decreased activity at higher concentrations. 6. Some amino acids can act as energy sources for the incorporation. 7. The effect of increasing the concentration of labelled amino acid is different for different amino acids, and complex effects occur on the addition of amino acid mixtures. 8. It is concluded that the amino acid incorporation into protein finally obtained is the result of many interacting factors related to the structural and metabolic state of the mitochondrion.
| S-EPMC1264760 | biostudies-other
Project description:Amino acid analyses of mitochondrial membranes are compared with the amino acid composition of whole mitochondria (Alberti, 1964) and found to be very similar except in the cystine content. The composition of the endogenous amino acids found in freshly prepared mitochondria has been established and shown to differ considerably from the amino acid composition of membranes or whole mitochondria. The amino acids produced during anaerobic incubation of mitochondria at pH7.4, on the other hand, resemble the membrane in composition, supporting the view that neutral proteinase activity is responsible for their appearance. Aerobic incubation produces a similar pattern of amino acids except that amino acids such as proline, serine, asparagine, glutamic acid and glutamine, which can be metabolically utilized under aerobic conditions, are present to a smaller extent. The presence of large relative concentrations of endogenous taurine, cysteic acid and oxidized glutathione and the accumulation of taurine during incubation is found. The selective retention of taurine and cysteic acid within the mitochondria is established. It is proposed that the first step in the degeneration of isolated mitochondria results from lipid hydroperoxide accumulation caused by the lack of glutathione reductase in isolated mitochondria.
| S-EPMC1177828 | biostudies-other
Project description:1. Incorporation of [(14)C]leucine into protein by isolated rat liver mitochondria was examined by using incubation media similar to those used by Sandell, Löw & Decken (1967) (medium A) and Roodyn, Reis & Work (1961) (medium B). The incorporation process was found to be almost completely inhibited in medium A. 2. By decreasing the amount of sucrose and omitting tris-hydrochloric acid from medium A, incorporation proceeded at a rate higher than that found in medium B. It was found that the inhibitory action of medium A was due to its high osmolarity. 3. Oxidative phosphorylation and RNA synthesis by the isolated mitochondria proceeded at the same rate in media essentially the same as media A and B. 4. There was a partial inhibitory action of medium A on leucine uptake by the mitochondria and also on the formation of leucyl-transfer-RNA. The major block of inhibition by the hyperosmolarity of medium A seemed to be located at a later step of protein synthesis involving mitochondrial ribosomes. 5. Protein synthesis by Escherichia coli B was only slightly inhibited, if at all, in hyperosmotic media in which protein synthesis by isolated mitochondria was completely stopped.
| S-EPMC1187594 | biostudies-other
Project description:1. Normal and thyroidectomized rats were treated with near-physiological doses of tri-iodothyronine. Liver mitochondria were isolated and incubated with radioactive amino acids. In normal rats tri-iodothyronine caused only a slight stimulation of incorporation into mitochondrial protein, but in thyroidectomized animals the incorporation was doubled. 2. There was a lag period of about 36 hr. after injection and the maximum effect was observed after 2 days. 3. Direct addition of tri-iodothyronine to the incubation medium had no effect on mitochondrial incorporation. 4. The incorporation was not due to bacterial, nuclear, lysosomal or microsomal contamination and the labelled particles had sedimentation properties identical with those of mitochondria, as followed by suitable enzyme markers. 5. Thyroid hormone treatment did not cause any marked alterations in the pattern of labelling of submitochondrial fractions and in all cases the most radioactive protein was in an insoluble lipid-rich fraction. The amino acid compositions of the total mitochondrial protein and the more radioactive lipoprotein were also unaltered. 6. Increases in the content of RNA and various cytochromes per mg. of mitochondrial protein were observed after treatment with tri-iodothyronine. These occurred slightly later than the stimulation of amino acid incorporation. 7. No uncoupling of oxidative phosphorylation was observed and the ATP production per mg. of mitochondrial protein increased. 8. It was concluded that tri-iodothyronine stimulated amino acid incorporation into mitochondrial protein and that the result is consistent with the view that treatment with thyroid hormone results in an enhanced selective synthesis of mitochondrial respiratory units.
| S-EPMC1206597 | biostudies-other
Project description:Ribosomal protein synthesis results in the genetically programmed incorporation of amino acids into a growing polypeptide chain. Faithful amino acid incorporation that accurately reflects the genetic code is critical to the structure and function of proteins as well as overall proteome integrity. Errors in protein synthesis are generally detrimental to cellular processes yet emerging evidence suggest that proteome diversity generated through mistranslation may be beneficial under certain conditions. Cumulative translational error rates have been determined at the organismal level, however codon specific error rates and the spectrum of misincorporation errors from system to system remain largely unexplored. In particular, until recently technical challenges have limited the ability to detect and quantify comparatively rare amino acid misincorporation events, which occur orders of magnitude less frequently than canonical amino acid incorporation events. We now describe a technique for the quantitative analysis of amino acid incorporation that provides the sensitivity necessary to detect mistranslation events during translation of a single codon at frequencies as low as 1 in 10,000 for all 20 proteinogenic amino acids, as well as non-proteinogenic and modified amino acids. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.
| S-EPMC5524608 | biostudies-literature
Project description:Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.
| S-EPMC1173765 | biostudies-other
Project description:Photoactivatable fluorophores are emerging optical probes for biological applications. Most photoactivatable fluorophores are relatively large in size and need to be activated by ultraviolet light; this dramatically limits their applications. To introduce photoactivatable fluorophores into proteins, recent investigations have explored several protein-labeling technologies, including fluorescein arsenical hairpin (FlAsH) Tag, HaloTag labeling, SNAPTag labeling, and other bioorthogonal chemistry-based methods. However, these technologies require a multistep labeling process. Here, by using genetic code expansion and a single sulfur-for-oxygen atom replacement within an existing fluorescent amino acid, we have site-specifically incorporated the photoactivatable fluorescent amino acid thioacridonylalanine (SAcd) into proteins in a single step. Moreover, upon exposure to visible light, SAcd can be efficiently desulfurized to its oxo derivatives, thus restoring the strong fluorescence of labeled proteins.
| S-EPMC8011588 | biostudies-literature
Project description:Livers of genetically obese Zucker rats showed, compared with lean controls, hypertrophy and enrichment in triacylglycerols, indicating that fatty acid metabolism was directed towards lipogenesis and esterification rather than towards fatty acid oxidation. Mitochondrial activities of cytochrome c oxidase and monoamine oxidase were significantly lower when expressed per g wet wt. of liver, whereas peroxisomal activities of urate oxidase and palmitoyl-CoA-dependent NAD+ reduction were unchanged. Liver mitochondria were able to oxidize oleic acid at the same rate in both obese and lean rats. For reactions occurring inside the mitochondria, e.g. octanoate oxidation and palmitoyl-CoA dehydrogenase, no difference was found between both phenotypes. Total carnitine palmitoyl-, octanoyl- and acetyl-transferase activities were slightly higher in mitochondria from obese rats, whereas the carnitine content of both liver tissue and mitochondria was significantly lower in obese rats compared with their lean littermates. The carnitine palmitoyltransferase I activity was slightly higher in liver mitochondria from obese rats, but this enzyme was more sensitive to malonyl-CoA inhibition in obese than in lean rats. The above results strongly suggest that the impaired fatty acid oxidation observed in the whole liver of obese rats is due to the diminished transport of fatty acids across the mitochondrial inner membrane via the carnitine palmitoyltransferase I. This effect could be reinforced by the decreased mitochondrial content per g wet wt. of liver. The depressed fatty acid oxidation may explain in part the lipid infiltration of liver observed in obese Zucker rats.
| S-EPMC1147245 | biostudies-other
Project description:Lanthipeptides represent a large class of cyclic natural products defined by the presence of lanthionine (Lan) and methyllanthionine (MeLan) cross-links. With the advances in DNA sequencing technologies and genome mining tools, new biosynthetic enzymes capable of installing unusual structural features are continuously being discovered. In this study, we investigated an O-methyltransferase that is a member of the most prominent auxiliary enzyme family associated with class I lanthipeptide biosynthetic gene clusters. Despite the prevalence of these enzymes, their function has not been established. Herein, we demonstrate that the O-methyltransferase OlvSA encoded in the olv gene cluster from Streptomyces olivaceus NRRL B-3009 catalyzes the rearrangement of a highly conserved aspartate residue to a ?-amino acid, isoaspartate, in the lanthipeptide OlvA(BCSA). We elucidated the NMR solution structure of the GluC-digested peptide, OlvA(BCSA)GluC, which revealed a unique ring topology comprising four interlocking rings and positions the isoaspartate residue in a solvent exposed loop that is stabilized by a MeLan ring. Gas chromatography-mass spectrometry analysis further indicated that OlvA(BCSA) contains two dl-MeLan rings and two Lan rings with an unusual ll-stereochemistry. Lastly, in vitro reconstitution of OlvSA activity showed that it is a leader peptide-independent and S-adenosyl methionine-dependent O-methyltransferase that mediates the conversion of a highly conserved aspartate residue in a cyclic substrate into a succinimide, which is hydrolyzed to generate an Asp or isoAsp containing peptide. This overall transformation converts an ?-amino acid into a ?-amino acid in a ribosomally synthesized peptide, via an electrophilic intermediate that may be the intended product.
| S-EPMC6812601 | biostudies-literature
Project description:In the mitochondrial genome of the hemichordate Balanoglossus carnosus, the codon AAA, which is assigned to lysine in most metazoans but to asparagine in echinoderms, is absent. Furthermore, the lysine tRNA gene carries an anticodon substitution that renders its gene product unable to decode AAA codons, whereas the asparagine tRNA gene has not changed to encode a tRNA with the ability to recognize AAA codons. Thus, the hemichordate mitochondrial genome can be regarded as an intermediate in the process of reassignment of mitochondrial AAA codons, where most metazoans represent the ancestral situation and the echinoderms the derived situation. This lends support to the codon capture hypothesis. We also show that the reassignment of the AAA codon is associated with a reduction in the relative abundance of lysine residues in mitochondrial proteins.
| S-EPMC19900 | biostudies-literature