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81.
Summary A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA). 相似文献
82.
Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly 13C15N-labeled proteins. In-phase 13C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from 13C to 1H leads to E.COSY-type cross peaks, from which the 3JH
co coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin. 相似文献
83.
Nuclear magnetic resonance studies of the location and function of plant nutrients in vivo 总被引:2,自引:1,他引:1
The cytoplasmic and vacuolar pools of ammonium, inorganic phosphate and potassium can be studied non-invasively in plant tissues using high resolution nuclear magnetic resonance spectroscopy. The techniques that allow these pools to be discriminated in vivo are described and their application to plants is reviewed with reference to the phosphorus, nitrogen and potassium nutrition of root tissues. 相似文献
84.
Josefa Salgado Jose Villalain Juan C. Gómez-Fernández 《European biophysics journal : EBJ》1993,22(2):151-155
-Tocopherol, ubiquinone-10 and ubiquinol-10 have been studied by high resolution magic angle samples spinning 13C-nuclear magnetic resonance in egg yolk phosphatidylcholine multilamellar vesicles model membranes in order to assess their location and the induced perturbations on this model system. -Tocopherol is placed in such a position that it is in close contact with the head group of the phospholipid and exposed to the solvent. In this position it significantly perturbs the phospholipid head group, the 5a-CH3 and the 7a-CH3 groups being the closest parts to the membrane surface. On the other hand, ubiquinol-10 perturbs the membrane surface more than ubiquinone-10, but neither compound significantly changed the phospholipid head group conformation. 相似文献
85.
NMR imaging of root water distribution in intact Vicia faba L plants in elevated atmospheric CO2 总被引:1,自引:1,他引:0
The effect of elevated atmospheric CO2 on water distribution in the intact roots of Vicia faba L. bean seedlings grown in natural soil was studied noninvasively with proton (1H) nuclear magnetic resonance (NMR) imaging. Exposure of 24-d-old plants to atmospheric CO2-enriched air at 650 cm3 m?3 produced significant increases in water imaged in upper roots, hypogeal cotyledons and lower stems in response to a short-term drying-stress cycle. Above ground, drying produced negligible stem shrinkage and stomatal resistance was unchanged. In contrast, the same drying cycle caused significant depletion of water imaged in the same upper root structures in control plants subject to ambient CO2 (350 m3 m?3), and stem shrinkage and increased stomatal resistance. The results suggest that inhibition of transpiration caused by elevated CO2 does not necessarily result in attenuation of water transport from lower root structures. Inhibition of water loss from upper roots and lower stem in elevated CO2 environments may be a mitigating factor in assessing deleterious effects of greenhouse changes on crops during periods of dry climate. 相似文献
86.
Ronnitte S. Badar-Goffer Nicola M. Thatcher Peter G. Morris Herman S. Bachelard 《Journal of neurochemistry》1993,61(6):2207-2214
(1) The energy state and free intracellular calcium concentration ([Ca2±i) of super-fused cortical slices were measured in moderate hypoxia (~65 μM O2), in mild hypoglycaemia (0.5 mM glucose), and in combinations of the two insults using 19F and 31P NMR spectroscopy. (2) Neither hypoxia nor hypoglycaemia alone caused any significant change in [Ca2±i. Hypoxia caused a 40% fall in phosphocreatine (PCr) content but not in ATP level, and hypoglycaemia produced a slight fall in both (as expected from previous studies). These changes in the energy state recovered on return to control conditions. (3) A combined sequential insult (hypoxia, followed by hypoxia plus hypoglycaemia) produced a 100% increase in [Ca2±, and a decrease in PCr level to ~25% of control. The reverse combined sequential insult (hypoglycaemia, followed by hypoglycaemia plus hypoxia) had the same effect. On return to control conditions there was some decrease in [Ca2±i and a small increase in PCr content, but neither recovered to control levels. (4) Exposure of the tissue to the combined simultaneous insult (hypoxia plus hypoglycaemia) immediately after the control spectra had been recorded resulted in a fivefold increase in [Ca2±i and a similar decrease in PCr level to 20–25% of control. There was little if any change of [Ca2±i or PCr level on return to control conditions. (5) These results are discussed in terms of metabolic adaptation of some but not all of the cortical cells to the single type of insult, which renders the tissues less vulnerable to the combined insult. 相似文献
87.
Abstract: Ischemia-induced changes in 31 P NMR relaxation were examined in 16 piglets. NMR spectra were acquired under control conditions and during complete cerebral ischemia induced via cardiac arrest. Changes in T 1 were assessed directly in six animals during control conditions and after 30–45 min of complete ischemia when changes in brain P1 levels had reached a plateau. The T 1 for P1 did not change, i.e., 2.3 ± 0.5 s during control conditions versus 2.4 ± 1.0 s during ischemia. To evaluate phosphocreatine and ATP, two types of spectra, with a long (25-s) or short (1-s) interpulse delay time, were collected during the first 10 min of ischemia (n = 10). Both types of spectra showed the same time course of changes in phosphocreatine and ATP levels, implying that the T 1 relaxation times do not change during ischemia. There were no changes in the linewidths of phosphocreatine, ATP, or P1 during ischemia, implying that the T *2 values remain constant. Our results suggest that the 31 P T 1 and T *2 for phosphocreatine, Pi , and ATP do not change during ischemia, and therefore changes in 31 P NMR peak intensity accurately reflect changes in metabolite concentrations. 相似文献
88.
Comparison of glucose fermentation by suspended and gel-entrapped yeast cells: An in vivo nuclear magnetic resonance study 总被引:1,自引:0,他引:1
Phosphorus-31 nuclear magnetic resonance ((31)P NMR) was used to compare the anaerobic metabolism of glucose by suspended and gel-entrapped Saccharomyces bayanus cells. The fermentation of glucose was carried out in a reaction system with continuous circulation through the NMR sample tube. The intracellular pH and the levels of some phosphorylated compounds were the levels of some phosphorylated compounds were noninvasively monitored by (31)P NMR while glucose, fermentation products, and biomass were determined by analytic techniques comparisons showed that no significant differences are observed in the relative concentrations in the spectra, but distinct profiles for the variation of both intracellular and extracellular pH are found. The internal pH of immobilized cells is maintained at a constant value throughout the fermentation as opposed to freely suspended cells for which a steady decrease in the internal pH occurs. A faster and stronger acidification is also observed in the external medium of the assays with suspended cells. Furthermore, higher yields for ethanol and biomass production and lower yields of fermentation by-products are obtained with immobilized cells. It is concluded that the higher intracellular pH achieved in the presence of the gel matrix had a regulatory effect on the metabolism which favored the ethanol production pathway. (c) 1993 John Wiley & Sons, Inc. 相似文献
89.
Tyrosyl ring motions in alpha-lytic protease were investigated by solid-state deuterium nuclear magnetic resonance (NMR) spectroscopy in lyophilized enzyme powder, in powder suspended in organic solvents, and in aqueous crystals. Ring flipping rates were determined by examining deuterium quadrupole echo line shapes. Of the four Tyr residues in the enzyme, one was flipping at the slow (< or =10(3) s(-1)) and one at the fast (> or =10(7) s(-1)) exchange limit of the line shape experiment in all the environments tested. Flipping rates of the remaining two Tyr residues depended markedly on the solvent, with the lowest flipping rates (< or =10(3) s(-1) for both residues) observed in the enzyme powder, whether dry or suspended in hydrophobic tert-butyl methyl ether. In hydrophilic dioxane and acetonitrile, the mobility of these residues increased to 10(4) and 10(5) s(-1). The latter rate rose further to 10(6) s(-1) in the hydrated hydrophilic solvents and to > or =10(7) s(-1) in aqueous crystals. The deuterium spectrum of native alpha-lytic protease was compared with that of the enzyme whose active center was covalently modified with an inhibitor, which binds next to Tyr-123, constraining its ring. This experiment revealed that water addition to acetonitrile specifically increased the flipping rate of this active center residue. Librational motions ("wobbling"), estimated by their effect on spin-lattice relaxation times, were slowest in the anhydrous solvents, intermediate in the hydrated solvents, and fastest in the aqueous crystals. Thus, alpha-lytic protease is more rigid in organic solvents than in water, as judged by mobility of its tyrosyl residues. Water stripping by hydrophilic solvents did not increase enzyme rigidity, nor were there clear correlations between mobility and either enzymatic activity or solvent dielectric constant. 相似文献
90.
The complete sequence-specific assignment of resonances in the1H-NMR spectrum of the polypeptide neurotoxin III (Hm III) from the sea anemoneHeteractis macrodactylus is described. Comparison of the chemical shifts and pattern of NOEs for Hm III with those for the related toxin Hp III fromHeteractis paumotensis, which differs only in the substitution of Asn for Tyr at position 11, shows that the overall secondary and tertiary structures are conserved. The largest differences in chemical shift caused by the substitution at position 11 are observed for the NH resonances of Arg-13, Thr-14, Ala-15, Leu-17, and Cys-26. The CH resonances influenced most are those of ASP-6, Gly-9, Leu-17, and Glu-42, while the most affected CH resonances are from Leu-17, Glu-28, and Lys-32. The absence of long-range NOEs to the aromatic ring of Tyr-11 as well as the lack of significant chemical shift effects on residues outside the loop comprising residues 7–16 confirm that this part of the loop makes no long-lived contacts with the rest of the molecule. The deviations from random coil shifts of Hm III are compared with those of the related anemone toxins Hp II, Hp III, and toxin I fromStichodactyla helianthus (Sh I). The similarity in deviations in chemical shift as a function of sequence position for these four toxins emphasizes the overall structural homology among these polypeptides. 相似文献