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Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures. 总被引:4,自引:2,他引:2 下载免费PDF全文
Protein phosphorylation was demonstrated in Bradyrhizobium japonicum bacteroids in vivo and in cultures in vivo and in vitro. Comparison of in vivo-labeled phosphoproteins of bacteroids and of cultured cells showed differences in both the pattern and intensity of labeling. In cultured cells, comparison of the labeling patterns and intensities of in vivo- and in vitro-labeled phosphoproteins showed a number of similarities; however, several phosphoproteins were found only after one of the two labeling conditions. The labeling intensity was time dependent in both in vivo and in vitro assays and was dependent on the presence of magnesium in in vitro assays. Differences in the rates of phosphorylation and dephosphorylation were noted for a number of proteins. The level of incorporation of 32P into protein was only 2% or less of the total phosphate accumulated during the in vivo labeling period. Several isolation and sample preparation procedures resulted in differences in labeling patterns. Phosphatase inhibitors and several potential metabolic effectors had negligible effects on the phosphorylation pattern. There were no significant changes in the phosphorylation patterns of cells cultured on mannitol, acetate, and succinate, although the intensity of the labeling did vary with the carbon source. 相似文献
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Protein Synthesis by Bradyrhizobium japonicum Bacteroids Declines as a Function of Nodule Age 下载免费PDF全文
Isolated bacteroids of Bradyrhizobium japonicum accumulated exogenously supplied [(sup35)S]methionine or [(sup3)H]leucine and incorporated them into cytosolic proteins. The accumulation of these labeled amino acids was inhibited by azide. Only 3 to 6% of these accumulated amino acids were incorporated into protein. Protein synthesis was not stimulated by incubation of bacteroids in the presence of potassium salts, malate, or amino acids, but azide, chloramphenicol, and acridine did inhibit the process. No prominent differences were observed in autoradiograms after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of (sup35)S-labeled bacteroid proteins as a function of nodule age. The rates of protein synthesis and protein turnover declined during nodule development. Protein synthesis declined about 60% between 14 and 20 days after planting, which is the period of a rapid increase in acetylene reduction activity. This correlation suggests a metabolic mechanism by which significant amounts of cellular energy are diverted to the nitrogen fixation process. 相似文献
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Investigation of the H(2) Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids 总被引:5,自引:0,他引:5 下载免费PDF全文
The H2-oxidizing complex in Rhizobium japonicum 122 DES bacteroids failed to catalyze, at a measurable rate, 2H1H exchange from a mixture of 2H2 and 1H2 in presence of 2H2O and 1H2O, providing no evidence for reversibility of the hydrogenase reaction in vivo. In the H2 oxidation reaction, there was no significant discrimination between 2H2 and 1H2, indicating that the initial H2-activation step in the over-all H2 oxidation reaction is not rate-limiting. By use of improved methods, an apparent Km for H2 of 0.05 micromolar was determined. The H2 oxidation reaction in bacteroids was strongly inhibited by cyanide (88% at 0.05 millimolar), theonyltrifluoroacetone, and other metal-complexing agents. Carbonyl cyanide m-chlorophenylhydrazone at 0.005 millimolar and 2,4-dinitrophenol at 0.5 millimolar inhibited H2 oxidation and stimulated O2 uptake. This and other evidence suggest the involvement of cytochromes and nonheme iron proteins in the pathway of electron transport from H2 to O2. Partial pressures of H2 at 0.03 atmosphere and below had a pronounced inhibitory effect on endogenous respiration by bacteroid suspensions. The inhibition of CO2 evolution by low partial pressures of H2 suggests that H2 utilization may result in conservation of oxidizable substrates and benefits the symbiosis under physiological conditions. Succinate, acetate, and formate at concentrations of 50 millimolar inhibited rates of H2 uptake by 8, 29, and 25%, respectively. The inhibition by succinate was noncompetitive and that by acetate and formate was uncompetitive. A concentration of 11.6 millimolar CO2 (initial concentration) in solution inhibited H2 uptake by bacteroid suspensions by 18%. Further research is necessary to establish the significance of the inhibition of H2 uptake by succinate, acetate, formate, and CO2 in the metabolism of the H2-uptake-positive strains of Rhizobium. 相似文献
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Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-beta-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis. 相似文献
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RNA interference in mammalian cells by chemically-modified RNA 总被引:24,自引:0,他引:24
RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo. 相似文献
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Efficient and selective recognition of DNA by proteins is due to sequence-specific interactions with a target site and nonselective electrostatic interactions that promote the target's rapid location. If synthetic molecules could mimic these functions, they would render a wide range of chromosome sequences accessible to rationally designed probes. Here we describe conjugates between bispeptide nucleic acids (bisPNAs) designed to specifically recognize duplex DNA and peptides that have been designed to promote rapid sequence recognition. Peptide design was based on the surface of staphylococcal nuclease, a cationic DNA binding protein with low sequence selectivity. We observe that attachment of the designed peptide increases rates of strand invasion by 100-fold relative to unmodified bisPNA. The peptide can contain D-amino acids, increasing the likelihood that it will be stable in cell extract and inside cells. Binding of the conjugate containing the D-amino acid peptide occurred over a broad range of experimental conditions and was sensitive to a single mismatch. Strand invasion was efficient at neutral to basic pH, a wide range of temperatures (0-65 degrees C), and in the presence of up to 7 mM Mg(2+) and 100 mM Na(+) or K(+). Our data suggest that attachment of peptides that mimic cationic protein surfaces to PNAs can afford conjugates that mimic the rapid and selective binding that characterizes native DNA binding proteins. Rapid strand invasion over a wide range of experimental conditions should further expand the utility of strand invasion by PNAs. 相似文献
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Isocitrate dehydrogenase of Bradyrhizobium japonicum is not required for symbiotic nitrogen fixation with soybean 下载免费PDF全文
A mutant strain of Bradyrhizobium japonicum USDA110 lacking isocitrate dehydrogenase activity was created to determine whether this enzyme was required for symbiotic nitrogen fixation with soybean (Glycine max cv. Williams 82). The isocitrate dehydrogenase mutant, strain 5051, was constructed by insertion of a streptomycin resistance gene cassette. The mutant was devoid of isocitrate dehydrogenase activity and of immunologically detectable protein, indicating there is only one copy in the genome. Strain 5051 grew well on a variety of carbon sources, including arabinose, pyruvate, succinate, and malate, but, unlike many microorganisms, was a glutamate auxotroph. Although the formation of nodules was slightly delayed, the mutant was able to form nodules on soybean and reduce atmospheric dinitrogen as well as the wild type, indicating that the plant was able to supply sufficient glutamate to permit infection. Combined with the results of other citric acid cycle mutants, these results suggest a role for the citric acid cycle in the infection and colonization stage of nodule development but not in the actual fixation of atmospheric dinitrogen. 相似文献