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1.
The synthesis of glutathione peroxidase from [75Se]selenite was studied in slices and cell-free extracts from rat liver. The incorporation of [75Se]selenocysteine at the active site was detected by carboxymethylation and hydrolysis of partially purified glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) in the presence of [3H]selenocysteine and subsequent amino acid analysis. The synthesis of glutathione peroxidase in slices was inhibited by cycloheximide or puromycin and 75Se was incorporated from [75Se]selenite into free selenocysteine and selenocysteyl tRNA. Increasing concentrations of selenocystine caused a progressive dilution of the 75Se and a corresponding decrease in glutathione peroxidase labeling. In cell-free systems, [75Se]selenocysteyl tRNA was the best substrate for glutathione peroxidase synthesis. These results indicate the existence in rat liver of the de novo synthesis of free selenocysteine and a translational pathway of selenocysteine incorporation into glutathione peroxidase  相似文献   

2.
1. [14C]Malonyl-CoA was incorporated into isoprenoids by cell-free yeast preparations, by preparations from pigeon and rat liver, and by Hevea brasiliensis latex. 2. In agreement with previous reports the incorporation of acetyl-CoA into isoprenoids was not inhibited by avidin and was not stimulated by HCO3. In a cell-free yeast preparation addition of HCO3 stimulated the formation of fatty acids from acetyl-CoA and decreased the incorporation into unsaponifiable lipids. 3. The labelling patterns of β-hydroxy-β-methylglutaryl-CoA formed from [2-14C]- and [1,3-14C]-malonyl-CoA in rat and pigeon liver preparations were those that would be expected if malonyl-CoA underwent decarboxylation to acetyl-CoA before incorporation. 4. The labelling pattern of ergosterol formed by cell-free yeast preparations from [2-14C]malonyl-CoA was also consistent with decarboxylation of malonyl-CoA before incorporation. 5. The incorporation of [2-14C]malonyl-CoA into mevalonate by rat liver preparations was related to the malonyl-CoA decarboxylase activity present in the preparation.  相似文献   

3.
The chemical synthesis of 24,25-dihydro[32-14C]lanosterol is described. The incubation of this material with a cell-free system from Saccharomvoes cerevisiae or with a microsomal preparation from rat liver resulted in both cases in the release of [14C]formic acid. This result suggests that in the biosynthesis of ergosterol in yeast, as well as in that of cholesterol in higher animals, the 14α-methyl group of lanosterol is removed as formic acid. In both systems, the measurement of the rate of release of [14C]formic acid from 24,25-dihydro[32-14C]lanosterol provides a simple and direct assay of lanosterol 14α-demethylase. Carbon monoxide inhibited both yeast and liver 14α-demethylase.  相似文献   

4.
The utilization of [1-14C]hexadecyl-[2-3H]ethyleneglycol and [1-14C]hexadecyl-[2-3H]glycerol as substrates for acyltransferase, phosphotransferase, phosphorylcholine, and phosphorylethanolamine transferase, and O-alkyl cleavage activities in cell-free preparations from normal rat liver and preputial gland tumors of mice was investigated. Our studies demonstrate that alkylethyleneglycols, like alkylglycerols, can serve as substrates for acyltransferases in both the liver and tumor microsomes; the product alkylacylethyleneglycerol can be readily deacylated by pancreatic lipase. A polar lipid was formed from the alkylethyleneglycol by the tumor homogenates in the presence of ATP and Mg2+; although the small quantities formed precluded absolute identification, its thin-layer Chromatographic behavior in acidic and basic solvent systems indicated that a free phosphate group was present. As expected, phosphorylbase transferases in these preparations did not utilize either the alkylethyleneglycol or alkylglycerol as substrates. The O-alkyl moiety of hexadecyl-ethyleneglycol was oxidized to hexadecanal by a tetrahydropteridine-dependent cleavage enzyme in rat liver microsomes, whereas in the tumor microsomes this activity was not present. We conclude that alkylethyleneglycols are metabolized in a manner similar to alkylglycerols and perhaps by identical enzymes.  相似文献   

5.
The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [75Se]selenocysteine and [75Se]selenite as substrates. [75Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [75Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [35S]cysteme, the most highly purified 75Se-fractions were > 100-fold purified relative to 35S. These fractions contained < 0.7% of the [35S]cysteine originally present in the total tRNA. When [35Se]selenocysteyl tRNA was purified from a mixture of 14C-labeled amino acids, over 97% of the [14C]aminoacyl tRNA was removed. The [75Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [75Se]selenocysteine, reaction with hydroxylamine and recovery of [75Se]selenocysteyl hydroxamic acid and release of 75Se by ribonuclease. The specificity of [75Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system.  相似文献   

6.
The branched-chain 2-oxo acids which accumulate in maple-syrup-urine disease inhibited the production of acetylcholine and of lipids, proteins, nucleic acids and of CO2. in sliced adult rat brains incubated with [U-14C] glucose. Inhibition of the biosynthetic reactions was proportional to the inhibition of CO2 production, even though the flux of radioactivity into the biosynthetic products was less than 2% of that to CO2. The oxo acids reduced the production of 14CO2, from [U-14C] glucose and from [2-14C]pyruvic acid more than from [1-14C]pyruvic acid in sliced brains. They inhibited the solubilized oxoglutarate dehydrogenase complex more than they did the solubilized pyruvate dehydrogenase complex. Valine and isoleucine, which also accumulate in maple-syrup-urine disease, inhibited pyruvate kinase from rat brain allosterically. Quantitative comparison of the effects of the disease metabolites on cell-free systems with their effects on fluxes in intact cells indicated that the inhibition of oxoglutarate dehydrogenase appeared to be functionally significant. The residual activities of the other enzymes studied were adequate to support the normal flux of carbohydrates. The oxo acids were effective at concentrations within the range reported to occur in patients with maple-syrup-urine disease. The effects on biosyntheses including that of acetylcholine would be expected to impair brain development and function and could be important in the development of brain disease in the patients. In contrast to the results with metabolites from maple-syrup-urine disease, metabolites which accumulate in phenylketonuria (phenylalanine and 2-oxo-3-phenylpropionic acid) did not inhibit carbohydrate utilization or the biosynthetic reactions studied, under the conditions of these experiments.  相似文献   

7.
Formyl-[35S]methionine is incorporated into histones synthesized by a mouse ascites cell-free system supplemented with histone mRNA and f-[35S]met-tRNAf from yeast. Most of the [35S]methionine incorporated can be shown to be at the N-terminus by Edman degradation after deformylation. This indicates that methionine can be used as the initiator amino acid for histones in this cell-free system.  相似文献   

8.
Cell-free extracts from leaves of Tanacetum vulgare synthesised geraniol and nerol (3,7-dimethylocta-trans-2-ene-1-ol and its cis isomer) in up to 11·9 and 2·4% total yields from IPP-[4-14C] and MVA-[2-14C] respectively. Optimum preparations were obtained from plant material just before the onset of flowering. The ratio of the monoterpenols varied 28-fold for different preparations under conditions where these products or their phosphate esters were not interconverted. Similar extracts incorporated α-terpineol-[14C] and terpinen-4-ol-[14C] (p-menth-1-en-8- and -4-ol respectively) in 0·05 to 2·2% yields into a compound tentatively identified as isothujone (trans-thujan-3-one), and preparations from flowerheads converted IPP-[4-14C] in 2·7% yield into geranyl and neryl β-d-glucosides. Inhibitors of IPP-isomerase had little effect on the incorporation of IPP into the monoterpenols in cell-free systems from which endogenous compounds of low molecular-weight had been removed. The inference that a pool of protein-bonded DMAPP or its biogenetic equivalent was present was supported by the demonstration that geraniol and nerol biosynthesised in the absence of the inhibitors were predominantly (65 to 100%) labelled in the moiety derived from IPP.  相似文献   

9.
—The conversion of [l-14C]palmitic acid to [1-14C]hexadecanol has been demonstrated with a cell-free system from developing rat brain. ATP, Coenzyme A and Mg2+ were required for the activity. Fatty aldehyde was found to be an intermediate in this reaction. The conversion of fatty acid to fatty alcohol was mainly localized in the microsomal fraction and the formation of hexadecanol showed absolute specificity towards NADPH while fatty aldehyde was formed even in the absence of exogenous reduced pyridine nucleotides. The brain microsomes showed maximal activity with stearic acid and the activities with palmitic and oleic acids were 65% and 38% respectively of that with stearic acid. This enzymic reduction increased with age and showed a maximum in the 15-day old rat brain.  相似文献   

10.
Aged discs cut from Kennebec potato tubers were inoculated with one of the following: an elicitor preparation from mycelia of Phytophthora infestans race 4, zoospores from either race 4 or race TY complex of this fungus, or sodium arachidonate. At 24 hr intervals after inoculation, four successive 0.5 mm thick layers of tissue were cut from the discs. This tissue was analysed for accumulated phytoalexins and also used to prepare cell-free enzyme systems for lubimin biosynthesis. In tissue treated with either the elicitor preparation or race 4 zoospores, levels of phytoalexin accumulation were highest in the first layer of tissue. Surprisingly, however, cell-free lubimin biosynthesis from [1-14C]isopentenyl pyrophosphate was also generally greater in preparations derived from the first 0.5 mm of tissue. Accumulation of phytoalexins in tissue inoculated with zoospores from race TY complex was very low, whereas cell-free biosynthetic activity was initially comparable to that seen in preparations from tissue treated with the elicitor preparation. By the end of the experimental period lower layers of tissue from discs treated with sodium arachidonate contained the highest levels of phytoalexins and yielded cell-free enzyme preparations with the greatest lubimin biosynthetic activity.  相似文献   

11.
Abstract— cell-free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [14C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble 14C-labeled proteins formed in the cell-free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of 14C-labeled amino acids. Criteria for the formation of vinblastine-binding, 14C-labeled proteins were: (1) aggregation of 14C-labeled soluble protein by one mm -vinblastine sulfate and (2) immunoprecipitation of 14C-labeled soluble protein by an antiserum against vinblastine sulfate-precipitable material. Criteria for the formation of [3H]colchicine-binding, 14C-labeled protein were based upon: (1) co-precipitation of the 3H-and 14C-labeled materials by vinblastine sulfate and (2) the coincidence of 3H- and 14C-labeled elution peaks from columns of Sephadex G-200, DEAE-Sephadex A-50 and isoelectric focusing. Both in the in vitro and in the in vivo system, 14C-labeled amino acids were incorporated into soluble proteins of the post-microsomal supernatant fraction. Proteins labeled with 14C-labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, 14C-labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G-200 which is identical to [3H]colchicine-binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine-binding and colchicine-binding proteins in the in vitro cell-free system.  相似文献   

12.
The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of polyhydroxyalkanoate by Rhodococcus ruber from succinic acid was investigated using nuclear magnetic resonance analysis. Polymer produced from [2,3-13C]- and [1,4-13C]succinate showed that the C-1-C-2 and C-4-C-5 fragments of 3HV were derived from carbons 2 and 3 of succinate, essentially without bond cleavage, and carbon 3 of 3HV was derived from a carboxyl carbon of succinate. Using [1,2-13C]succinate it was demonstrated that the C-1-C-2 bond of succinate was cleaved during polymer biosynthesis. Methylmalonyl-coenzyme A (CoA) mutase activity was detected in cell-free extracts of R. ruber by enzyme assay and HPLC analysis of reaction products. A pathway, involving the known methylmalonyl-CoA pathway for propionate formation in Propionibacteria, followed by the established pathway for PHA biosynthesis from propionyl-CoA and acetyl-CoA, is proposed for the biosynthesis of 3HV from succinate by R. ruber. Correspondence to: A. J. Anderson  相似文献   

13.
《Phytochemistry》1987,26(2):331-334
Both [3H]thymidine and [3H]deoxyadenosine were found to be incorporated into the nuclear DNA of wheat embryos immediately after dry embryos were allowed to imbibe aqueous solutions of the radioactive precursors. The early labelled DNA sedimented in a manner suggesting that replicative intermediates were already formed within the first 90 min of germination. However, aphidicolin remained without any effect on this early DNA synthesis. Likewise, a cell-free system derived from early embryos incorporated [3H]dCTP into DNA independently of the presence of aphidicolin. On the contrary, dideoxyTTP inhibited the DNA synthesis considerably. It is concluded that a proportion of the resting wheat embryo cells is able to initiate a replicative DNA synthesis immediately upon imbibition. The synthesis seems, however, to proceed with the participation of a γ-like, rather than an α-like, DNA polymerase.  相似文献   

14.
Summary Cell-free protein synthesizing systems were prepared from the livers of chick embryos at selected ages and the characteristics of individual fractions were compared. While polysomes showed decreasing size with older embryos, isolated polysomes did not differ significantly in amino acid incorporating activity when assayed with standard cell sap. When assayed with standard polysomes, cell sap activity decreased with increasing developmental age whether incorporation was measured using [3H]lysine, [3H]leucine, or [3H]aminoacyl-tRNA. Free amino acid concentrations in the cell sap showed reproducible independent variation during development which was taken into consideration in calculating net amino acid incorporation. A large increase in ribonuclease activity was observed during development; however, nuclease inhibitor activity was absent before day 15 but increased thereafter. Aminoacyl-tRNA synthetase activity did not vary significantly. It is proposed that the observed changes in the rate of cell-free protein synthesis result not only from increasing ribonuclease activity with increasing developmental age but also from changes in the activity of other soluble factors.This is paper VI in a series; paper V is reference 6. The series title is based on earlier work with systems derived from fowl which synthesized two genetic variants of serum albumin21.This research was supported in part by a grant from the Damon Runyon Memorial Fund (DRG-1125). Dr. H. M. Jernigan was an N.I.H. Postdoctoral Fellow (5 F02 GM 50944-02).To whom all inquiries are to be addressed.  相似文献   

15.
The 30000 g supernatants from cell-free extracts of Nepeta cataria leaf tissue and leaf callus tissue have mevalonic acid kinase, mevalonic acid phosphate kinase and mevalonic acid pyrophosphate decarboxylase activities. The callus tissue cell-free extract produced mevalonic acid pyrophosphate and isopentenyl pyrophosphate; however, very little mevalonic acid phosphate was observed. The leaf cell-free extracts incubated with [14C]-mevalonic acid produced higher amounts of mevalonic acid phosphate. When both the leaf cell-free extract and the callus cell-free extract were incubated with [14C]-mevalonic acid in the presence of iodoacetamide, the ion exchange column elution profile was cleaner, which was confirmed by PC. Apparently the callus tissue 30000 g supernatant contains mevalonic acid phosphorylating enzymes even though there is no production of the methyl cyclopentane monoterpenes.  相似文献   

16.
Distinct sets of cellular proteins were labeled with [3H]myristic and [3H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [3H]myristate originating from [3H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.  相似文献   

17.
A cell-free system derived from seed embryos of barley (Hordeum vulgare cv. Zephyr) grain has been used to prepare substrate quantities of radioactively-labelled C5-C20 intermediates of terpenoid biosynthesis. The purification and characterization of high specific activity all-trans farnesyl-[4, 8, 12-3H] and all-trans geranylgeranyl-[4, 8, 12, 16-3H] pyrophosphates, suitable for use in studies of sterol and carotenoid biosynthesis, are described in detail. The effects of the plant growth retardant AMO 1618 on the system are reported.  相似文献   

18.
Summary 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 in-sensitive to cycloheximide. Poly(U) addition stimulated [14C]phenylalanine incorporation by the preincubated submitochondrial system. Upon the addition of 7.5S mRNA that was iso-lated 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.  相似文献   

19.
The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [35S]methionine-containing chymotryptic peptides of the 52,000 Mr polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [35S]methionine in vivo. From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 Mr product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.  相似文献   

20.
Ricin A chain was radioactively labeled using reductive alkylation, lactoperoxidase catalyzed iodination, and reaction with iodoacetamide or N-ethylmaleimide (NEM). The inhibition of cell-free rat liver protein synthesis by the modified A chains and the ribosome binding characteristics of each of the labeled derivatives was examined. [3H] NEM was found to quantitatively react with the A chain sulfhydryl group normally involved in a disulfide bond with the B chain in intact ricin. Labeling the protein with [3H] NEM had no effect on the in vitro inhibition of protein synthesis by the A chain. [3H] NEM-labeled A chain binds to rat liver ribosomes in a manner which is dependent on the concentrations of NaCl and Mg2+. At optimal Mg2+ concentration (5.5 mM), A chain binding to ribosomes is saturable and fully reversible either by dilution of the reaction mixture or by addition of unlabeled A chain. At 5.5 mM Mg2+, A chain was found to bind to a single site on rat liver ribosomes with a dissociation constant of 6.2 X 10?8 M. [3H] NEM-labeled A chain did not bind to isolated 40S ribosomal subunits and bound to 60S ribosomal subunits with a 1 : 1 molar stoichiometry and a dissociation constant of 2.2 X 10?7 M. The relationship between ribosome binding and A chain inhibition of eucaryotic protein synthesis is discussed.  相似文献   

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