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11.
B. C. Loughman 《Plant and Soil》1987,99(1):63-74
Summary Little is known about the biochemical basis of the genotypic differences in the capacity for ion absorption and transport
shown by many crop species. If these differences reflect the abundance of a specific membrane component or the activity of
an enzyme we need to have some indication of thein vivo operation of these systems in whole plants. Thein vivo assessment of glycolytic enzymes is illustrated by the effects of mannose on the transport of phosphate in maize varieties.
The application of high resolution31P-NMR to the study of intermediary metabolismin vivo is also helpful in following transport capacity.
The five-fold rise in respiratory rate that occurs when freshly cut potato slices are maintained in aerated water for 24 hours
is accompained by the turning on of a wide range of biochemical systems. Major increases in the capacity for absorption of
phosphate from low concentrations (0.1 μM–10 μM) and in the phosphorylative ability of the tissue are seen, indicating the synthesis of a carrier involved in phosphate transport.
These capacities differ markedly between individual tissues of the tuber,i. e. pith, parenchyma, cortex and buds and large differences have been observed between comparable tissue from different varieties.
Varieties grown under similar conditions have been compared and shown to exhibit different kinetics with respect to the development
of the low concentration absorption site and in their sensitivity to the effects of uncouplers such as 2,4-dinitrophenol. 相似文献
12.
A reagent (I, N4-(9-fluorenylmethyloxycarbonyl-4-amino-1-oxyl-4-succinimidyloxycarbonyl-2,2,6,6-tetramethylpiperidine)) that acylates calmodulin specifically at lysines 75 and 148 was recently described (Jackson and Puett, 1984). Chromatographic procedures are described that permit purification to apparent homogeneity of a 1 : 1 and a 2 : 1 adduct characterized by modification at just Lys 75 or at Lys 75 and Lys 148, respectively. These adducts are suitable for detailed characterization in an effort to provide information on calmodulin structure-function relationships. The adducts were incapable of, or exhibited low potency (e.g., 0.1% that of calmodulin) in, stimulating the activity of an activatable bovine brain cyclic nucleotide phosphodiesterase (3,5-cyclic AMP 5-nucleotidehydrolase, EC 3.1.4.17) preparation. Electron paramagnetic resonance (EPR) spectroscopy of the adducts yielded rotational correlation times of approximately 3–6 nsec, in agreement with the expected value for a hydrated protein of this molecular weight (5–7 nsec). Thus, the nitroxide reporter group appears to monitor closely the motion of the protein, and there is no evidence of a major conformational change in the derivative relative to calmodulin. Interestingly, removal of the fluorenylmethyloxycarbonyl portion from the 1 : 1 adduct to give a deprotected 1 : 1 adduct resulted in apparent greater mobility of the probe, since the rotational correlation coefficient was found to be 1 nsec. Circular dichroic spectra were obtained over the wavelength interval 200–250 nm on the two adducts and on the deprotected 1 : 1 adduct. These derivatives, like calmodulin, exhibited a Ca2+-mediated increase in helicity, and the spectra of the adducts in the presence of a chelating agent and in the presence of saturating Ca2+ were similar to those obtained for calmodulin. Thus, the adducts have secondary structures similar to the native protein. Proton nuclear magnetic resonance spectra were determined in the aromatic region (6–8 ppm) for the deprotected 1 : 1 adduct before and after reduction of the nitroxide with ascorbate. The nitroxide had little effect on the chemical shifts of the two tyrosines and the single histidine relative to calmodulin, although the histidine C4 resonance was markedly altered by the addition of ascorbate. In order to explore in greater detail the tertiary structure of the 1 : 1 adduct, a reagent similar to I, but not paramagnetic, was synthesized. This compound II, -N-(9-fluorenylmethyloxycarbonyl)alanine N-hydroxysuccinimide ester, like I, forms a 1 : 1 adduct at Lys 75 and a 2 : 1 adduct at Lys 75 and Lys 148. Proton NMR spectra of adducts with II were not complicated by the relaxation effects arising from adducts with I; thus more definitive assignments could be made to the upfield resonances, including the fluorene protons. Again, it was possible to conclude that adduct formation had no major effect on the tertiary structure of the protein as monitored by chemical shifts associated with various residues. We conclude that modification of just Lys 75, a residue in the long connecting helix of calmodulin, does not lead to major changes in protein conformation but does interfere with the ability of calmodulin to stimulate an activatable form of bovine brain cyclic nucleotide phosphodiesterase. 相似文献
13.
J. Sianoudis A. C. Küsel A. Mayer L. H. Grimme D. Leibfritz 《Archives of microbiology》1987,147(1):25-29
P-31 NMR investigations were performed with the green alga Chlorella fusca under anaerobic conditions in the dark and in the light.In spectra of cells in the dark the signal of intracellular, nonvacuolar Pi indicates a pH in its chemical environment of 7.0–7.2. Upon illumination this signal looses intensity and shifts to lower field, corresponding to a pH of 7.7. Further downfield no other signal that could be attributed to a Pi-pool in more alkaline environment was detected. By the use of 2-deoxyglucose-6-phosphate as an indicator of cytoplasmic pH, this Pi-signal was assigned to the cytoplasm. The pH increase in the cytoplasm upon transfer of cells from the dark to the light is the same as that previously observed upon transfer of cells from anaerobic to aerobic conditions.In cells performing only cyclic photophosphorylation the cytoplasmic pH is lower than in photosynthesizing cells but still 0.2 pH units higher than in the cells in the dark. The reasons for the missing of a signal of stromal Pi and for the difference in cytoplasmic pH in photosynthesizing cells and those capable only of cyclic photophosphorylation are discussed.Non-standard abbreviations 2dG
2-Deoxyglucose
- dG-6-P
2-deoxyglucose-6-phosphate
- DCMU
3,4-dichlorophenyl-dimethylurea
- MOPSO
3-(N-morpholino)-2-hydroxypropane sulfonic acid
- P-31 NMR
P-31 nuclear magnetic resonance 相似文献
14.
Norma J. Greenfield Mussadeq Hussain John Lenard 《Biochimica et Biophysica Acta (BBA)/General Subjects》1987,926(3)
The vacuoles of logarithmic and stationary stage cells were compared by 31P-NMR with regard to pH, orthophosphate (Pi) content and average size of polyphosphate. The vacuoles of stationary cells had lower pH higher Pi content, and polyphosphates of longer average chain lenght, although total polyphosphate content was about the same as in logarithmic cells. The lower vacuolar pH in stationary cells was the major cause of a larger cytoplasmic-vacuolar pH gradient. Addition of NH4Cl, (NH4)2SO4, methylamine or amantadine at pH 8 to cells in either stage caused an icnrease in both cytoplasmic and vacuolar pH, with little or no change in the cytoplasmic-vacuolar pH gradient. However, the administration of ammonium salts to the cells at pH 8.0 resulted in rapid hydrolysis of the intravacuolar polyphosphate to tripolyphosphate and Pi, with attendant redistribution of Pi between the vacuolar and cytoplasmic compartments. 相似文献
15.
The internal pH of peroxisomes in the yeasts Hansenula polymorpha, Candida utilis and Trichosporon cutaneum X4 was estimated by 31P nuclear magnetic resonance (NMR) spectroscopy. 31P NMR spectra of suspensions of intact cells of these yeasts, grown under conditions of extensive peroxisomal proliferation, displayed two prominent Pi-peaks at different chemical shift positions. In control cells grown on glucose, which contain very few peroxisomes, only a single peak was observed. This latter peak, which was detected under all growth conditions, was assigned to cytosolic Pi at pH 7.1. The additional peak present in spectra of peroxisome-containing cells, reflected Pi at a considerably lower pH of approximately 5.8–6.0. Experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazon (CCCP) and the ionophores valinomycin and nigericin revealed that separation of the two Pi-peaks was caused by a pH-gradient across a membrane separating the two pools. Experiments with chloroquine confirmed the acidic nature of one of these pools. In a number of transfer experiments with the yeast H. polymorpha it was shown that the relative intensity of the Pi-signal at the low pH-position was correlated to the peroxisomal volume fraction. These results strongly suggest that this peak has to be assigned to Pi in peroxisomes, which therefore are acidic in nature. The presence of peroxisome-associated Pi was confirmed cytochemically.Abbreviations CCCP
Carbonyl cyanide m-chlorophenylhydrazon
- DCCD
N,N-dicyclohexylcarbodiimide 相似文献
16.
A. C. Kuesel W. Kuhn J. Sianoudis L. H. Grimme D. Leibfritz A. Mayer 《Archives of microbiology》1989,151(5):434-438
The possibility to apply N-15 in vivo NMR spectroscopy to study algal N-metabolism has been investigated. N-15 labelled cells
of the green alga Chlorella fusca, subjected to nitrogen starvation and N-14 labelled cells supplied with K15NO3 after prolonged nitrogen starvation were monitored by N-15 in vivo NMR spectroscopy at different times after the change in
their nitrogen supply. During 20–40 min, necessary for the acquisition of 1 spectrum, the cells were under dark anaerobic
conditions, but the relative amounts of the metabolites detected did not change. Signals from 2 acid amides, from the side
chain nitrogens of arginine and lysine, from prolin as well as 4 signals from α amino groups of amino acids were detected.
Besides two signals not yet reported in the literature were found. They may be due to amino compounds, but not to amino acids.
The amount of free amino acids in the cells increases not only upon resupply of nitrogen starved cells with nitrate but also
during the first hours after nitrate depletion. The spectra obtained from N-15 labelled autospores show that N-15 in vivo
NMR spectroscopy can be applied to the investigation of N metabolism of the cells. 相似文献
17.
Polysaccharides excreted by cowpea Rhizobium strains JLn(c) and RA-1 were mixtures of complex acidic exopolysaccharides and low molecular weight neutral glucans. These polymers were fractionated using gel filtration chromatography. Purified fractions of the acidic heteropolymer reacted with peanut agglutinin to give precipitin bands when subjected to Ouchterlony gel diffusion. The acidic exopolysaccharide was found to contain mainly glucose, galactose, glucuronic acid, mannose and fucose. The non-carbohydrate substituents of the acidic heteropolymer were pyruvate, acetate and uronate which were identified by infrared and proton nuclear magnetic resonance spectroscopy as well as by chemical analysis.Abbreviations EPS
Extracellular polysaccharide
- YEM
yeast extract mannitol
- PNA
peanut agglutination
-
1H-NMR
proton nuclear magnetic resonance 相似文献
18.
Franz-Georg Hanisch Jasna Peter-Katalinic Heinz Egge Ursula Dabrowski Gerhard Uhlenbruck 《Glycoconjugate journal》1990,7(6):525-543
O-Linked glycans were isolated from human skim milk mucins or mucin-derived high-molecular weight glycopeptides and fractionated by anion exchange chromatography into neutral and acidic alditols. Major oligosaccharides contained in the acidic fraction were purified by high performance liquid chromatography and structurally characterized by a combination of fast atom bombardment mass spectrometry, methylation analysis and 500 MHz 1H-nuclear magnetic resonance spectroscopy. The structural aspects exhibited by these major species in the acidic fraction resemble those established previously for the neutral oligosaccharides from human skim milk mucins: 1) the size of the alditols varies from tri- to decasaccharides, 2) the core structure is of the ubiquitous type 2, 3) the backbone sequences are of the poly-N-acetyllactosamine type with a particular preponderance of linearly extended GlcNAc beta(1-3)Gal (major) or GlcNAc beta(1-6)Gal units (minor). 相似文献
19.
Calluses induced fromPterocladia capillacea have been kept in culture for more than three years. They exhibit a fast growth rate, owing to the release of single cells, which in turn develop into new callus. The effect of various media and culture conditions upon growth was investigated. In order to confirm the identity of the callus cells, a 0,45 mg incoculum was grown that yielded 15 g dried callus within six weeks. Polysaccharides from this material (5.5 g) were analysed by13C NMR spectroscopy. This produced a spectrum typical of agar and very similar to the one obtained for agar extracted fromP. capillacea plants. However, the callus agar displayed no gel-forming properties, even after alkali modification.author for correspondence 相似文献
20.
Summary
1H-NMR spectra of bovine pancreatic trypsin inhibitor (BPTI) both native and oxidized by chloramine T, are reported. The spectrum of the oxidized form is characterized by the appearance of two singlets for methyl group shifted 0.60 and 0.46 ppm downfield with respect to the native form. 相似文献