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51.
Transgenic barley plants overexpressing a 13-lipoxygenase to modify oxylipin signature 总被引:1,自引:0,他引:1
Sharma VK Monostori T Göbel C Hänsch R Bittner F Wasternack C Feussner I Mendel RR Hause B Schulze J 《Phytochemistry》2006,67(3):264-276
Three chimeric gene constructs were designed comprising the full length cDNA of a lipoxygenase (LOX) from barley (LOX2:Hv:1) including its chloroplast targeting sequence (cTP) under control of either (1) CaMV35S- or (2) polyubiquitin-1-promoter, whereas the third plasmid contains 35S promoter and the cDNA without cTP. Transgenic barley plants overexpressing LOX2:Hv:1 were generated by biolistics of scutella from immature embryos. Transformation frequency for 35S::LOX with or without cTP was in a range known for barley particle bombardment, whereas for Ubi::cTP-LOX no transgenic plants were detected. In general, a high number of green plantlets selected on bialaphos became yellow and finally died either in vitro or after potting. All transgenic plants obtained were phenotypically indistinguishable from wild type plants and all of them set seeds. The corresponding protein (LOX-100) in transgenic T0 and T1 plants accumulated constitutively to similar levels as in the jasmonic acid methyl ester (JAME)-treated wild type plants. Moreover, LOX-100 was clearly detectable immunocytochemically within the chloroplasts of untreated T0 plants containing the LOX-100-cDNA with the chloroplast target sequence. In contrast, an exclusive localization of LOX-100 in the cytoplasm was detectable when the target sequence was removed. In comparison to sorbitol-treated wild type leaves, analysis of oxylipin profiles in T2 progenies showed higher levels of jasmonic acid (JA) for those lines that displayed elevated levels of LOX-100 in the chloroplasts and for those lines that harboured LOX-100 in the cytoplasm, respectively. The studies demonstrate for the first time the constitutive overexpression of a cDNA coding for a 13-LOX in a monocotyledonous species and indicate a link between the occurrence of LOX-100 and senescence. 相似文献
52.
Putrescine carbamoyltransferase (PTCase) catalyzes the conversion of carbamoylputrescine to putrescine and carbamoyl phosphate (CP), a substrate of carbamate kinase (CK). The crystal structure of PTCase has been determined and refined at 3.2 Å resolution. The trimeric molecular structure of PTCase is similar to other carbamoyltransferases, including the catalytic subunit of aspartate carbamoyltransferase (ATCase) and ornithine carbamoyltransferase (OTCase). However, in contrast to other trimeric carbamoyltransferases, PTCase binds both CP and putrescine with Hill coefficients at saturating concentrations of the other substrate of 1.53 ± 0.03 and 1.80 ± 0.06, respectively. PTCase also has a unique structural feature: a long C‐terminal helix that interacts with the adjacent subunit to enhance intersubunit interactions in the molecular trimer. The C‐terminal helix appears to be essential for both formation of the functional trimer and catalytic activity, since truncated PTCase without the C‐terminal helix aggregates and has only 3% of native catalytic activity. The active sites of PTCase and OTCase are similar, with the exception of the 240′s loop. PTCase lacks the proline‐rich sequence found in knotted carbamoyltransferases and is unknotted. A Blast search of all available genomes indicates that 35 bacteria, most of which are Gram‐positive, have an agcB gene encoding PTCase located near the genes that encode agmatine deiminase and CK, consistent with the catabolic role of PTCase in the agmatine degradation pathway. Sequence comparisons indicate that the C‐terminal helix identified in this PTCase structure will be found in all other PTCases identified, suggesting that it is the signature feature of the PTCase family of enzymes Proteins 2012; © 2012 Wiley Periodicals, Inc. 相似文献
53.
Cell biology of molybdenum in plants and humans 总被引:1,自引:0,他引:1
The transition element molybdenum (Mo) needs to be complexed by a special cofactor in order to gain catalytic activity. With the exception of bacterial Mo-nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor Moco, which in different variants is the active compound at the catalytic site of all other Mo-containing enzymes. In eukaryotes, the most prominent Mo-enzymes are nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and the mitochondrial amidoxime reductase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also requires iron, ATP and copper. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco-carrier/binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms. In humans, Moco deficiency is a severe inherited inborn error in metabolism resulting in severe neurodegeneration in newborns and causing early childhood death. This article is part of a Special Issue entitled: Cell Biology of Metals. 相似文献
54.
RF Martins V Zina EB Silva MT Rebelo E Figueiredo Z Mendel OS Paulo JC Franco SG Seabra 《Journal of genetics》2012,91(2):e75-e78
55.
Lehrke M Rump S Heidenreich T Wissing J Mendel RR Bittner F 《The Biochemical journal》2012,441(3):823-832
The Moco (molybdenum cofactor) sulfurase ABA3 from Arabidopsis thaliana catalyses the sulfuration of the Moco of aldehyde oxidase and xanthine oxidoreductase, which represents the final activation step of these enzymes. ABA3 consists of an N-terminal NifS-like domain that exhibits L-cysteine desulfurase activity and a C-terminal domain that binds sulfurated Moco. The strictly conserved Cys430 in the NifS-like domain binds a persulfide intermediate, which is abstracted from the substrate L-cysteine and finally needs to be transferred to the Moco of aldehyde oxidase and xanthine oxidoreductase. In addition to Cys?3?, another eight cysteine residues are located in the NifS-like domain, with two of them being highly conserved among Moco sulfurase proteins and, at the same time, being in close proximity to Cys?3?. By determination of the number of surface-exposed cysteine residues and the number of persulfide-binding cysteine residues in combination with the sequential substitution of each of the nine cysteine residues, a second persulfide-binding cysteine residue, Cys2??, was identified. Furthermore, the active-site Cys?3? was found to be located on top of a loop structure, formed by the two flanking residues Cys?2? and Cys?3?, which are likely to form an intramolecular disulfide bridge. These findings are confirmed by a structural model of the NifS-like domain, which indicates that Cys?2? and Cys?3? are within disulfide bond distance and that a persulfide transfer from Cys?3? to Cys2?? is indeed possible. 相似文献
56.
Heat intolerance: does gene transcription contribute? 总被引:2,自引:0,他引:2
57.
Disulfide bonds in soybean trypsin inhibitor (Kunitz) were simultaneously reduced and alkylated using tri-n-butylphosphine and 2-vinylquinoline at pH 7.6 in 0.11 M Tris-4.4 M urea, 41% ethanol. The resulting S-β-2-quinolylethylated protein (2-QE-STI) has a new absorption peak at 315–318 nm. Its quinoline fluorescence can be excited above 310 nm independently of intrinsic protein fluorescence. Free 2-quinolylethylcysteine (2-QEC) shows unexpectedly weak fluorescence. Quinoline absorption in 2-QEC and 2-QE-STI changes with pH. The apparentpK values determined spectrophotometrically are near 5 for 2-QEC and 3 for 2-QE-STI. Fluorescence decreased with increasing pH and in the presence of chloride ions. Both structural and charge effects thus appear to influence the absorption and fluorescence of the quinoline group. Corrected fluorescence emission (excited at 316 nm) of neutral 2-QE-STI diluted in 0.1 N H2SO4 was directly proportional to concentration in the range 0.4–8 μm 2-QEC. The 2-QEC content of the protein derivative determined by UV absorption at pH 1.5 was in agreement with the expected value of four residues per mole. Fluorescence measurements ofS-2-quinolylethylated proteins may be especially useful as a sensitive, specific assay for cyst(e)ine residues. 相似文献
58.
59.
C M Mendel 《The Journal of steroid biochemistry and molecular biology》1990,37(2):251-255
A rapid filtration assay employing dextran-coated charcoal as acceptor particles for free hormone was used to measure the rates of dissociation of dihydrotestosterone (DHT), testosterone (T), and estradiol (E2) from their binding proteins in human serum at 37 degrees C. Because measurements were begun after each hormone had fully (greater than 99%) dissociated from albumin, the observed rates of dissociation correspond to the rates of dissociation of the sex hormone-binding globulin (SHBG)-hormone complexes. The dissociation rate constants of the hormone-SHBG complexes were determined to be 0.016 +/- 0.001, 0.056 +/- 0.002, and 0.083 +/- 0.003 s-1 for DHT, T, and E2, respectively, corresponding to half-times of dissociation (t1/2) of 43, 12 and 8.4 s, respectively. The physiological significance of these findings can best be appreciated by comparing these t1/2 s with the capillary and sinusoidal transit times of various tissues (less than 1 s to approximately 10 s). 相似文献
60.
Conditions for assay of molybdenum cofactor in barley shoot extracts in the presence of molybdate (25 mM N2MoO4) and the sulphydryl-group protector, reduced glutathione (5 mM) were optimized. Both total Mo-cofactor (assayed after heat-treatment of cell-free extracts) and ‘free’ Mo-cofactor (assayed in untreated cell-free extracts) were assayed. Compared to control plants grown in the absence of an exogenous nitrogen source total Mo-cofactor levels increased around 70 % when plants were grown for 4 days in the presence of either 15 mM KNO3 or 15 mM NH4NO3. Growth in the presence of 15 mM (NH4)2SO4 did not affect the Mo-cofactor level. Very similar results were seen when plants were transferred to these nitrogen sources for 24 hr after previous growth in the absence of an exogenous nitrogen source. In contrast ‘free’ Mo-cofactor levels of both KNO3 and NH4NO3-treated plants were increased 2-3-fold over untreated controls. Growth in the presence of (NH4)2SO4 did not affect the ‘free’ Mo-cofactor level. 相似文献