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161.
Abstract: The role of voltage-sensitive Ca2+ channels in mediating Ca2+ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca2+ channels. Concurrent 31P/19F and 23Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca2+] ([Ca2+]i), and intracellular Na+ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na+ content; however, a substantial increase in [Ca2+]i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca2+]i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca2+]i seen during ischemia. These data demonstrate that voltage-gated Ca2+ channels do not mediate significant Ca2+ flux during ischemia in this cell line and suggest an important role for Ca2+i stores, the Na+/Ca2+ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca2+ level during ischemia. 相似文献
162.
Gordon C. K. Roberts 《Neurochemical research》1996,21(9):1117-1124
NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The
kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in
drug metabolism. Cytochromes P450 play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the
haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate
complexes. The other two enzymes, chloramphenicol acetyltransferase and β-lactamase, are responsible for bacterial resistance
to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme
A bound to the enzyme, while in the case of β-lactamase the pK of a specific lysine residue at the active site has been determined,
providing valuable information on the catalytic mechanism.
Special issue dedicated to Dr. Herman Bachelard. 相似文献
163.
Alessandro Pintar Meike Hensmann Kornelia Jumel Maureen Pitkeathly Stephen E. Harding Iain D. Campbell 《European biophysics journal : EBJ》1996,24(6):371-380
The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of1H-15N-correlated 2D HSQC,15N-edited 3D TOCSY-HMQC, and15N-edited 3D NOESY-HMQC spectra. By analysis of the -proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T1 and T2 relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.Abbreviations SH
Src homology
- GST
glutathione-S-transferase
- IPTG
isopropyl--D-galactopyranoside
- DTT
dithiothreitol
- PMSF
phenyl-methyl-sulphonyl-fluoride
- TBS
50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0
- MWCO
molecular weight cut off
- NMR
nuclear magnetic resonance
- HSQC
heteronuclear single-quantum correlation
- NOESY
nuclear Overhauser effect spectroscopy 相似文献
164.
Novel mono-O-acetylated GM3s, one containing 9-O-acetylN-glycolyl neuraminic acid and another containing 6-O-acetyl galactose, were isolated as a mixture from equine erythrocytes, and the structures were characterized by one- and two-dimensional proton nuclear magnetic resonance (NMR) and fast atom bombardment-mass spectrometry (FAB-MS). The position of theO-acetyl residue was identified by the downfield shift of the methylene protons at C-9 ofN-glycolyl neuraminic acid (9-O-Ac GM3) and C-6 of galactose (6-O-Ac GM3) in the NMR spectrum, in comparison to the respective non-acetylated counterparts. To confirm the presence of 6-O-Ac GM3, theO-acetylated GM3 mixture was desialylated withArthrobacter neuraminidase, giving 6-O-acetyl galactosyl glucosylceramide, the structure of which was estimated by NMR and FAB-MS, together with non-acetylated lactosylceramide with a ratio of 1:1.
Abbreviations: Ac, acetyl; Gc, glycolyl; NeuGc,N-Gc neuraminic acid; GM3 (Gc), GM3 containing NeuGc (II3NeuGc-LacCer); 4-O-Ac GM3 (Gc), GM3 containing 4-O-Ac NeuGc; 9-O-Ac GM3 (Gc), GM3 containing 9-O-Ac NeuGc; 6-O-Ac GM3 (Gc), GM3 containing 6-O-Ac Gal; 1D-NMR, one-dimensional nuclear magnetic resonance spectrometry; 2D-COSY, two-dimensional chemical shift-correlated spectrometry; FAB-MS, fast atom bombardment-mass spectrometry; GLC, gas-layer chromatography; GC-MS, gas chromatography-mass spectrometry; TLC, thin-layer chromatography; Ggl, ganglioside; Cer, ceramide; CMH, monohexosylceramide; LacCer, lactosylceramide; 6-O-Ac LacCer, LacCer containing 6-O-Ac Gal; Me2SO-d6,2H6-dimethylsufloxide; CMW, chloroform-methanol-water; Nomenclature and abbreviations of glycosphingolipids follow the system of Svennerholm (J Neurochem [1963]10: 613–23) and those recommended by the IUPAC-IUB Nomenclature Commission (Lipids [1977]12: 455–68). 相似文献
165.
Ernest V. Curto Ted T. Sakai Michael J. Jablonsky Sandrine Rio-Anneheim Jean-Claude Jacquinet N. Rama Krishna 《Glycoconjugate journal》1996,13(4):599-607
We present complete1H NMR assignments for two synthetic glycopeptides representative of the carbohydrateprotein linkage region of serglycin proteoglycans. The peptides are: Ser(Galp-Xylp)-Gly-Ser-Gly-Ser(Galp-Xylp)-Gly and, Ser(Galp-Xylp)-Gly-Ser(Galp-Xylp)-Gly-Ser(Galp-Xylp)-Gly. A number of 2D NMR spectra together with a 3D NOESY-TOCSY spectrum were acquired at 600 MHz to complete the assignments of the glycopeptides dissolved in water with 40% trifluoroethanol. Preliminary analysis of the NMR data suggests folded structures for the glycopeptides.A preliminary account of this work was presented at an International Symposium held at the University of Alabama at Birmingham in November, 1994 on the occasion of the 65th birthday of Professor Lennart Rodén. 相似文献
166.
The heterogeneous nature of microbial products as shown by solid-state13C CP/MAS NMR spectroscopy 总被引:1,自引:1,他引:0
Homoionic Na-, Ca-, and Al-clays were prepared from the <2 m fractions of Georgia kaolinite and Wyoming bentonite and mixed with sand to give artificial soils with 5, and 25% clay. The artificial soils were inoculated with microbes from a natural soil before incubation. Unlabelled and uniformly13C-labelled (99.9% atom) glucose were incorporated into the artificial soils to study the effects of clay types, exchangeable cations and clay contents on the mineralization of glucose-carbon and glucose-derived organic materials. Chemical transformation of glucose-carbon upon incorporation into microbial products and metabolites, was followed using solid-state13C CP/MAS NMR spectroscopy.There was a significant influence of exchangeable cations on the mineralization of glucose-carbon over a period of 33 days. At 25% clay content, mineralization of glucose-carbon was highest in Ca-soils and lowest in Al-soils. The influence of exchangeable cations on mineralization of glucose-carbon was more pronounced in soils with bentonite clay than those with kaolinite clay. Statistical analysis of data showed no overall effect of clay type on mineralization of glucose-carbon. However, the interactions of clay type with clay content and clay type with clay content and exchangeable cations were highly significant. At 25% clay content, the mineralization of glucose-carbon was significantly lower in Na- and Al-soils with Wyoming bentonite compared with Na- and Al-soils with Georgia kaolinite. For Ca-soils this difference was not significant. Due to the increased osmotic tension induced by the added glucose, mineralization of glucose-carbon was slower in soils with 5% clay than soils with 25% clay.Despite the differences in the chemical and physical characteristics of soils with Ca-, Na- and Al-clays, the chemical composition of organic materials synthesised in these soils were similar in nature. Assuming CP/MAS is quantitative, incorporation of uniformly13C-labelled glucose (99.9% atom) in these soils resulted in distribution of carbon in alkyl (24–25%), O-alkyl (56–63%), carbonyl (11–15%) and small amounts of aromatic and olefinic carbon (2–4%). However, as decomposition proceeded, the chemistry of synthesised material showed some changes with time. In the Ca- and Na-soils, the proportions of alkyl and carbonyl carbon decreased and that of O-alkyl carbon increased with time of incubation. However, the opposite trend was found for the Al-soil.Proton-spin relaxation editing (PSRE) subspectra clearly showed heterogeneity within the microbial products. Subspectra of the slowly-relaxing (long T1(H)) domains were dominated by alkyl carbon in long- and short-chain structures. The signals due to N-alkyl (55 ppm) and carbonyl carbon were also strong in these subspectra. These subspectra were very similar to those obtained for microbial and fungal materials and were probably microbial tissues attached to clay surfaces by polysaccharide extracellular mucilage. Subspectra of fast-relaxing (short T1(H)) domains comprised mostly O-alkyl and carbonyl carbon and were probably microbial metabolites released as neutral and acidic sugars into the extracellular environment, and strongly sorbed by clay surfaces. 相似文献
167.
The chemical and biological characteristics of humus within the Ah horizon (Calcic-Luvisol) have been studied. Attention was paid to variation in the NMR spectra of humic fractions and 13C values and to how these changes are related to different biological humic fraction activities.The chemical changes in particular involve the decrease of the aromatic component and the increase of the non-aromatic component within the horizon and the different 13C value not only within the horizon but also among the humic fractions distinctive of different molecular sizes.An attempt has been made to explain the vertical chemical changes in terms of processes affecting the biological characteristics of the high and low molecular size humic fractions. The main conclusions are that the low molecular size humic fractions, in the upper part of the horizon, are of greater importance with respect to the other humic fractions in influencing the enzyme activities linked to growth metabolism. The biological role of the high molecular size humic fractions characterised by a relevant content of peptidic- and carbohydratic-C is also presented. 相似文献
168.
Oscar Varela Patricia A. Zunszain Daniel O. Cicero Ricardo F. Baggio Daniel R. Vega María T. Garland 《Carbohydrate research》1996,280(2):187
The conformation in 2H2O of 4-thio-l-lyxono-1,4-lactone (1) was studied by nuclear magnetic resonance spectroscopy, by means of homonuclear (J1H,1H) and heteronuclear (J1H,13C) coupling constants. The couplings were directly measured by a two-dimensional heteronucleus-coupled ω1 hetero-half-filtered proton-proton correlation (HETLOC) experiment, which does not require 13C isotopic enrichment. In solution, the thiolactone ring of 1 adopts preferentially the E3 conformation, and its hydroxymethyl group populates mainly the gt rotamer. The X-ray diffraction data of a single crystal of 1 indicates that also in the solid state the thiolactone ring adopts an E3 conformation, with a puckering somewhat larger than that observed for aldono-1,4-lactones and furanose rings. The molecules are linked by hydrogen bonds, which form chains. Particularly, O-5 is fully engaged as donor and acceptor in hydrogen bonding and the rotameric conformation of the hydroxymethyl group of 1 is fixed in the tg form. 相似文献
169.
The Lys residues in the 75-residue Ca2+-binding protein calbindin D9k were reductively methylated with13C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca2+- and apo-forms of the protein, the13C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca2+-calbindin D9k and apo-calbindin D9k were obtained by combiningpH titration experiments and (1H,13C)-HMQC NMR spectroscopy. Each Lys residue in the Ca2+- and apo-forms of calbindin D9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D9k has a very similar structure compared to Ca2+-calbindin D9k, the removal of two Ca2+ ions from the protein leads to an increase of thepKa values of the Lys residues. 相似文献
170.
Sharfstein ST Tucker SN Mancuso A Blanch HW Clark DS 《Biotechnology and bioengineering》1994,43(11):1059-1074
Carbon-13 nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of a murine hybridoma cell line at two feed glutamine concentrations, 4.0 and 1.7 mM. Carbon-13 labeling patterns were used in conjunction with nutrient uptake rates to calculate the metabolic fluxes through the glycolytic pathway, the pentose shunt, the malate shunt, lipid biosynthesis, and the tricarboxylic acid (TCA) cycle. Decreasing the feed glutamine concentration significantly decreased glutamine uptake but had little effect on glucose metabolism. A significant incrase in antibody productivity occurred upon decreasing the feed glutamine level. The increased antibody productivity in concert with decreased glutamine uptake and no apparent change in glucolytic metabolism suggests that antibody production was not energy limited. Metabolic flux calculations indicate that (1) approximately 92% of the glucose consumed proceeds directly through glycolysis with 8% channeled through the pentose shunt; (2) lipid biosynthesis appears to be greater than malate shunt activity; and (3) considerable exchange occurs between TCA cycle intermediates and amino acid metabolic pools, leading to substantial loss of (13)C label from the TCA cycle. These results illustrate that (13)NMR spectroscopy is a powerfulf tool in the calculation of metabolic fluxes, particularly for exchange pathways where no net flux occurs. (c) 1994 John Wiley & Sons, Inc. 相似文献