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1.
An eight-nucleotide sequence in the potato virus X 3' untranslated region is required for both host protein binding and viral multiplication. 总被引:3,自引:1,他引:2
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Gel retardation and UV-cross-linking techniques were used to demonstrate that two tobacco proteins, with approximate molecular masses of 28 and 32 kDa, bind to a site within the 3' region of potato virus X (PVX) genomic RNA. The protein binding is specific, in that a 50-fold excess of unlabeled probe prevents formation of the complexes but no reduction is observed with a 2,000-fold molar excess of yeast tRNA. Complex formation is inhibited by poly(U) but is relatively unaffected by poly(A), poly(G), or poly(C-I). PVX RNA-host protein complex formation occurs in vitro at salt concentrations up to 400 mM. Deletion mapping indicates that the proteins bind within the 3' untranslated region (UTR) of PVX genomic RNA and that an 8-nucleotide U-rich sequence (5'-UAUUUUCU) is required for the binding. Deletion of the 8-nucleotide U-rich region from the 3' UTR of a sensitive PVX reporter virus that carries the luciferase gene in place of the PVX coat protein gene results in a more than 70,000-fold reduction in luciferase expression in tobacco protoplasts. RNA probes carrying the sequence GCGC in place of the central four contiguous uridines of the 8-nucleotide U-rich motif fail to bind host protein at detectable levels, and the same mutation, when introduced into the PVX reporter virus, eliminates viral multiplication. Mutations of 1 or 2 nucleotides within the same four uridines reduced both binding of host proteins and replication of reporter virus. These results indicate that the 8-nucleotide U-rich motif within the PVX 3' UTR is important for some aspect of viral multiplication and suggest that host protein binding plays a role in the process. 相似文献
2.
Michael H Woo John R Vance Ana R Otero Marcos Christian Bailly Mary-Ann Bjornsti 《The Journal of biological chemistry》2002,277(6):3813-3822
DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development. However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast. Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA complexes. Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722. The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1delta, rad52delta yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular target of R-3 and indicate that distinct drug-enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p. 相似文献
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The specificity of CTP:phosphocholine cytidylyltransferase from rat liver for phosphorylated bases has been investigated. The apparent Km for phosphocholine was 0.17 mM. As the number of methyl substituents on the phospho-base decreased, the apparent Km increased: 4.0 mM for phosphodimethylethanolamine, 6.9 for phosphomonomethylethanolamine and 68.4 for phosphoethanolamine. The Vmax for the reaction was similar for phosphocholine (12.6 mumol/min per mg protein), phosphomonomethylethanolamine (13.5 mumol/min per mg protein) and phosphoethanolamine (9.2 mumol/min per mg protein). When phosphodimethylethanolamine was the substrate, the Vmax was 3-fold higher (40.3 mumol/min per mg protein). Phosphoethanolamine, phosphomonomethylethanolamine and phosphodimethylethanolamine were competitive inhibitors of the cytidylyltransferase when phosphocholine was used as substrate with Ki values of 18.5 mM, 9.3 mM and 1.5 mM, respectively. The results show that the cytidylyltransferase is highly specific for phosphocholine. 相似文献
8.
Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules 总被引:1,自引:1,他引:0
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The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO2 fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O2. Phloemgirdling was effected 20 hours and exposure to Ar:O2 was effected 2 to 3 hours before initiation of experiments. Nodule and root CO2 fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O2 decreased nodule CO2 fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO2 fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to 14CO2. In contrast to nodules, roots exported very little radioactivity, and most of the 14C was exported as organic acids. The nonphotosynthetic CO2 fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO2 assimilation. Nodules fixed CO2 at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO2 fixation. The results of this study showed that exposure of nodules to Ar:O2 reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO2 fixation, nitrogenase activity, nodule-specific respiration, and transport of 14C photoassimilate to nodules. These results indicate that nodule CO2 fixation in alfalfa is associated with N assimilation. 相似文献
9.
Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the synthesis of phosphatidylcholine by the stepwise transfer of methyl groups from S-adenosylmethionine to the amino head group of PE. PE N-methyltransferase was solubilized from a microsomal membrane fraction of rat liver using the nonionic detergent Triton X-100 and purified to apparent homogeneity. Specific activities of PE N-methyltransferase with PE, phosphatidyl-N-monomethylethanolamine (PMME), and phosphatidyl-N,N-dimethylethanolamine (PDME) as substrates were 0.63, 8.59, and 3.75 mumol/min/mg protein, respectively. The purified enzyme was composed of a single subunit with a molecular mass of 18.3 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methylation activities dependent on the presence of PE, PMME, and PDME and the 18.3-kDa protein co-eluted when purified PE N-methyltransferase was subjected to gel filtration on Sephacryl S-300 in the presence of 0.1% Triton X-100. All three methylation activities eluted with a Stokes radius 2.1 A greater than that determined for pure Triton micelles (molecular mass difference of 27.4 kDa). Two-dimensional analysis of PE N-methyltransferase employing nonequilibrium pH gradient gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of a single isoform. Analysis of enzyme activity using PE, PMME, and PDME at various Triton X-100 concentrations indicated the enzyme follows the "surface dilution" model proposed for other enzymes that act at the surface of mixed micelle substrates. Initial velocity data for all three lipid substrates (at fixed concentrations of Triton X-100) were highly cooperative in nature. Hill numbers for PMME and PDME ranged from 3 at 0.5 mM Triton to 6 at 2.0 mM Triton. All three methylation activities had a pH optimum of 10. These results provide evidence that a single membrane-bound enzyme catalyzes all three methylation steps for the conversion of PE to phosphatidylcholine. 相似文献
10.
Interaction of human thrombospondin with types I-V collagen: direct binding and electron microscopy 总被引:14,自引:9,他引:5
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N J Galvin P M Vance V M Dixit B Fink W A Frazier 《The Journal of cell biology》1987,104(5):1413-1422
Binding of thrombospondin (TSP) to types I-V collagen was examined by direct binding assays using 125I-TSP and by visualization of rotary-shadowed intermolecular complexes in the electron microscope. The binding of TSP was highest to type V collagen in the absence of Ca, while lower but significant levels of binding were observed to all other collagen types in the presence or absence of Ca. Unlike intact TSP, the trimeric collagen-binding domain of TSP composed of 70-kD chains showed no Ca dependence in its binding to type V collagen. Further evidence for binding of TSP to types I and III collagen was obtained by competition studies in which these soluble collagens effectively inhibited binding of 125I-TSP to immobilized type V collagen. The binding of TSP to type V collagen was inhibited by heparin and fucoidin, both high-affinity ligands of TSP's heparin-binding domain. mAb A6.1, which binds to the 70-kD domain of TSP, is also the best of a panel of anti-TSP mAbs at inhibiting the TSP-collagen interaction. Electron microscopy of rotary-shadowed replicas of TSP-collagen complexes revealed that all five types of collagen examined had a binding site for TSP at one end of the pepsinized, triple helical molecule. The specificity of this site was tested by examining the ability of BSA to form a complex with the end of the pepsinized collagens. Rotary-shadowed replicas revealed a low frequency of apparent BSA-collagen complexes, and histograms of these data showed no evidence for the preferential association of BSA with the end of the collagen molecules. In addition to the specific end site, type V collagen had an internal binding site for TSP located about two-thirds of the distance along the length of the collagen molecule from the end site. The internal binding site for TSP on type V collagen is apparently the site responsible for the higher affinity binding of TSP to that protein observed in direct binding assays. The trimeric 70-kD collagen-binding domain of TSP bound to the same sites on the collagens as did intact TSP. 相似文献