Microsomal components in relation to amino acid incorporation by preparations from the developing rat brain.
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ABSTRACT:
SUBMITTER: Lim L
PROVIDER: S-EPMC1270565 | biostudies-other | 1967 Jul
REPOSITORIES: biostudies-other
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Project description:1. A study has been made of the ability of cerebral microsome-cell sap systems, taken from rats at various ages, to incorporate [(14)C]valine. 2. The systems appear to follow Michaelis-Menten kinetics when cell sap is considered as substrate, microsomes as enzyme and total counts/mg. of microsomal protein as a measure of reaction velocity. 3. Reproducible ;affinity constant' values are obtained, and the system from 4-day-old rats has a higher V(max.) and affinity constant than the system from adult rats. 4. It is suggested the the amino acid-incorporating ability of different systems may be compared by this means.
| S-EPMC1264992 | biostudies-other
Project description:Methods of genetic code manipulation, such as nonsense codon suppression and genetic code reprogramming, have enabled the incorporation of various nonproteinogenic amino acids into the peptide nascent chain. However, the incorporation efficiency of such amino acids largely varies depending on their structural characteristics. For instance, l-α-amino acids with artificial, bulky side chains are poorer substrates for ribosomal incorporation into the nascent peptide chain, mainly owing to the lower affinity of their aminoacyl-tRNA toward elongation factor-thermo unstable (EF-Tu). Phosphorylated Ser and Tyr are also poorer substrates for the same reason; engineering EF-Tu has turned out to be effective in improving their incorporation efficiencies. On the other hand, exotic amino acids such as d-amino acids and β-amino acids are even poorer substrates owing to their low affinity to EF-Tu and poor compatibility to the ribosome active site. Moreover, their consecutive incorporation is extremely difficult. To solve these problems, the engineering of ribosomes and tRNAs has been executed, leading to successful but limited improvement of their incorporation efficiency. In this review, we comprehensively summarize recent attempts to engineer the translation systems, resulting in a significant improvement of the incorporation of exotic amino acids.
| S-EPMC6386890 | biostudies-literature
Project description:1. The incorporation of (14)C-labelled amino acids into polysomal protein was studied in a system consisting of polysomes and pH5 enzyme obtained 4 and 40hr. after a single intraperitoneal injection of 4-dimethylamino-3'-methylazobenzene. Labelling of the polysome fraction of preparations of both the 4hr.-treated and 40hr.-treated rats was considerably higher than in the normal control. 2. In further experiments on protein synthesis by polysomes from azo-dye-treated rats, the effect of replacing pH5 enzyme with cell sap was studied. Incorporation of (14)C-labelled amino acids into polysomal protein was similar to that of the control. 3. Aggregate size of polysomes obtained from rats injected previously with 4-dimethylamino-3'-methylazobenzene was studied by sucrose-gradient centrifugation. Polysomes prepared at 4hr. after azo-dye administration contained a considerable amount of large aggregates (approx. 700s), whereas at 40hr. after administration of the azo-dye the amount of large aggregates was less than in the control. 4. Determination of the ultraviolet spectra of polysome preparations from both normal and azo-dye-treated rats revealed no difference between the preparations. On the other hand, the ultraviolet spectra of cell-sap fractions from the different preparations showed that there is a definite shift in the absorption maximum from 272mmu (normal) to 267mmu, 40hr. after treatment, with an intermediate value of 270mmu for the preparation from 4hr.-treated rats. The absorption minimum changes from 250mmu (normal) to 245mmu for the preparation from 40hr.-treated rats.
| S-EPMC1264907 | biostudies-other
Project description:Microsomal membrane preparations from the developing endosperm of castor bean (Ricinus communis) catalysed the transfer of oleate from [14C]oleoyl-CoA to phosphatidylcholine (PtdCho). In the presence of NADH, radioactive ricinoleate (12-hydroxyoctadec-9-enoate) was synthesized from [14C]oleate, and this was largely recovered in PtdCho and as free fatty acid. The addition of unlabelled ricinoleoyl-CoA to these incubation mixtures did not increase the low [14C]ricinoleate concentration found in the acyl-CoA fraction nor decrease the [14C]ricinoleate concentration in PtdCho and free fatty acid, and thus no evidence was obtained for a hydroxylation with oleoyl-CoA as a substrate. The addition of NADH, necessary for the formation of ricinoleate, caused a decrease of the total radioactivity in PtdCho with a corresponding increase in the amount of label in free ricinoleic acid. This increase was due to the action of a phospholipase A, which released ricinoleic acid but not oleic acid from PtdCho. Such a phospholipase activity, attacking ricinoleoyl-PtdCho but not oleoyl-PtdCho, was also demonstrated in microsomal preparations from developing cotyledons of safflower and oil-seed rape. An analysis of the acyl groups at different positions in microsomal PtdCho of castor bean showed that ricinoleate was almost entirely associated with position sn-2. Likewise the [14C]ricinoleate in [14C]PtdCho formed after incubations with microsomal preparations with NADH and [14C]oleoyl-CoA resided in position sn-2 with none in position sn-1. In contrast, the [14C]linoleate formed by desaturation of [14C]oleoyl-PtdCho was present at both positions. In the presence of ATP, CoA and Mg2+, the ricinoleate acid released from PtdCho was activated to ricinoleoyl-CoA. The ricinoleoyl-CoA was an efficient acyl donor in the acylation of glycerol 3-phosphate (Gro3P) to yield phosphatidic acid and triacylglycerols. In microsomal preparations incubated with an equimolar mixture of [14C]oleoyl-CoA and [14C]ricinoleoyl-CoA in the presence of Gro3P, only a minor amount of [14C]ricinoleate entered PtdCho, and this was believed to be via the exchange of phosphocholine groups between a diacylglycerol pool and the PtdCho. On the basis of our results, a scheme of ricinoleate formation and its incorporation into triacylglycerols in castor-bean endosperm is proposed.
| S-EPMC1130577 | biostudies-other
Project description:1. Washed guinea-pig cerebral-cortex mitochondria incorporate [(14)C]leucine into their protein at a rate comparable with the rates reported for liver or heart mitochondria only if the mitochondria are separated from myelin and nerve endings by density-gradient centrifugation. 2. The non-mitochondrial components (myelin and nerve endings) of brain mitochondrial preparations incorporated [(14)C]leucine at a negligible rate. 3. The mitochondria do not require an exogenous supply of energy or a full supply of amino acids to support the process. 4. The incorporation rate was linear up to 2hr. aerobic incubation at 30 degrees and was inhibited by chloramphenicol, only slightly by actinomycin D and not by penicillin or pretreatment with ribonuclease. The observed incorporation is considered to be unlikely to be due to contaminating cytoplasmic ribosomes or bacteria. 5. The process was also studied in mitochondrial preparations from rabbit cerebral cortex and spinal cord.
| S-EPMC1265103 | 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:The last step in triacylglycerols (TAG) biosynthesis in oil seeds, the acylation of diacylglycerols (DAG), is catalysed by two types of enzymes: the acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). The relative contribution of these enzymes in the synthesis of TAG has not yet been defined in any plant tissue. In the presented work, microsomal preparations were obtained from sunflower and safflower seeds at different stages of development and used in DGAT and PDAT enzyme assays. The ratio between PDAT and DGAT activity differed dramatically between the two different species. DGAT activities were measured with two different acyl acceptors and assay methods using two different acyl-CoAs, and in all cases the ratio of PDAT to DGAT activity was significantly higher in safflower than sunflower. The sunflower DGAT, measured by both methods, showed significant higher activity with 18:2-CoA than with 18:1-CoA, whereas the opposite specificity was seen with the safflower enzyme. The specificities of PDAT on the other hand, were similar in both species with 18:2-phosphatidylcholine being a better acyl donor than 18:1-PC and with acyl groups at the sn-2 position utilised about fourfold the rate of the sn-1 position. No DAG:DAG transacylase activity could be detected in the microsomal preparations.
| S-EPMC3664747 | biostudies-literature
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:Subcellular fractions containing microsomes prepared from rat livers homogenized in the absence of EDTA catalysed the oxidation of cholesterol to 7alpha-hydroxycholesterol, 7-oxocholesterol, 7beta-hydroxycholesterol and 5alpha-cholestane-3beta,5,6beta-triol. These reactions required native protein, molecular oxygen and NADPH. It is suggested that these compounds are formed by a peroxidation analogous to the peroxidation of fatty acids catalysed by liver microsomal preparations. Incubations of [4-(14)C]cholesterol with microsomal preparations from rat liver homogenized in the presence of EDTA gave 7alpha-hydroxy[(14)C]cholesterol as the main product. This reaction required molecular oxygen and NADPH, and was inhibited by CO. The mass of 7alpha-hydroxycholesterol formed during the incubation was measured by a double-isotope-derivative dilution procedure. This procedure was used to assay the activity of cholesterol 7alpha-hydroxylase and to measure low concentrations of endogenous 7alpha-hydroxycholesterol in liver.
| S-EPMC1173563 | biostudies-other
Project description:The developing seeds of Borago officinalis (common borage) accumulate a triacylglycerol oil that is relatively rich in the uncommon fatty acid gamma-linolenate (octadec-6,9,12-trienoic acid). Incubation of developing, whole, cotyledons with [14C]oleate and [14C]linoleate showed that the gamma-linolenate was synthesized by the sequential desaturation of oleate----linoleate----gamma-linolenate. Microsomal membrane preparations from the developing cotyledons contained an active delta 6-desaturase enzyme that catalysed the conversion of linoleate into gamma-linolenate. Experiments were designed to manipulate the [14C]linoleate content of the microsomal phosphatidylcholine. The [14C]linoleoyl phosphatidylcholine labelled in situ was converted into gamma-linolenoyl phosphatidylcholine in the presence of NADH. The substrate for the delta 6-desaturase in borage was, therefore, the linoleate in the complex microsomal lipid phosphatidylcholine, rather than, as in animals, the acyl-CoA. This was further confirmed in experiments that compared the specific radioactivity of the gamma-linolenate, in acyl-CoA and phosphatidylcholine, that was synthesized when [14C]linoleoyl-CoA was incubated with microsomal membranes, NADH and non-radioactive gamma-linolenoyl-CoA. The delta 6-desaturase was positionally specific and only utilized the linoleate in position 2 of sn-phosphatidylcholine. Analysis of the positional distribution of fatty acids in the endogenous microsomal sn-phosphatidylcholine showed that, whereas position 1 contained substantial linoleate, only small amounts of gamma-linolenate were present. The results shed further light on the synthesis of C18 polyunsaturated fatty acids in plants and in particular its relationship to the regulation of the acyl quality of the triacylglycerols in oilseeds.
| S-EPMC1147430 | biostudies-other