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51.
Summary A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in1H–15N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys75 through Thr79 located in the central helix of calmodulin, and for the C-terminal residues Ser147 and Lys148. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr110 through Glu114.Istituto Guido Donegani, Novara, Italy 相似文献
52.
Hydrogen exchange kinetics of nucleic acids: Double and triple helices with Hoogsteen-type basepairs
Yuzuru Hayashi Mamoru Nakanishi Masamichi Tsuboi Toshikazu Fukui Morio Ikehara Ichiro Tazawa Yasuo Inoue 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,698(1):93-99
The kinetics of hydrogen-tritium exchange reaction have been followed by a Sephadex technique of a double-helical poly(ribo-2-methylthio-adenylic acid)·poly(ribouridylic acid) complex with the Hoogsteen-type basepair. Only one hydrogen in every 2-methylthio-adenine·uracil basepair has been found to exchange at a measurably slow rate, 0.023 s?1 (at 0°C), which is, however, much greater than that for a double-helix with the Watson-Crick type A·U pair. The kinetics of hydrogen-tritium exchange were also examined by triple-helical poly(rU)·poly(rA)·poly(rU) which involves both the Watson-Crick and Hoogsteen basepairings. Here, three hydrogens in every U·A·U base triplet have been found to exchange at a relatively slow rate, 0.0116 s?1 (at 0°C). The kinetics of hydrogen-deuterium exchange reactions of these polynucleotide helices have also been followed by a stopped-flow ultraviolet absorption spectrophotometry at various temperatures. On the basis of these experimental results, the mechanism of the hydrogen exchange reactions in these helical polynucleotides was discussed. In the triple helix, the rate-determining process of the slow exchange of the three (one uracil-imide and two adenine-amino) hydrogens is considered to be the opening of the Watson-Crick part of the U·A·U triplet. This opening is considered to take place only after the opening of the Hoogsteen part of the triplet. 相似文献
53.
Coupling of Dopamine Oxidation (Monoamine Oxidase Activity) to Glutathione Oxidation Via the Generation of Hydrogen Peroxide in Rat Brain Homogenates 总被引:16,自引:6,他引:10
Howard S. Maker Cipora Weiss Demetra J. Silides Gerald Cohen 《Journal of neurochemistry》1981,36(2):589-593
Abstract: Homogenates of perfused rat brain generated oxidized glutathione from reduced glutathione during incubation with dopamine or serotonin. This activity was blocked by pargyline. a monoamine oxidase inhibitor, or by catalase, a scavenger of hydrogen peroxide. These results demonstrate formation of hydrogen peroxide by monoamine oxidase and the coupling of the peroxide to glutathione peroxidase activity. Oxidized glutathione was measured fluorometrically via the oxidation of NADPH by glutathione reductase. In the absence of added dopamine or serotonin, a much smaller amount of reduced glutathione was oxidized: this activity was blocked by catalase, but not by pargyline. Therefore, endogenous production of hydrogen peroxide, not linked to monoamine oxidase activity, was present. These results indicate that glutathione peroxidase (linked to hexose monophosphate shunt activity) can function to eliminate hydrogen peroxide generated by monoamine oxidase and other endogenous sources in aminergic neurons. 相似文献
54.
Summary Oxygen uptake, carbon dioxide evolution and nitrogenase activity, measured either as hydrogen evolution (under argon 80%, oxygen 20%) or as the reduction of acetylene to ethylene, were assayed over the same time period by a direct mass-spectrometric method. When carbon dioxide evolution was used to estimate carbohydrate consumption, the results agreed with other work on whole plants. The RQ values obtained in these experiments were always less than 1.0 and thus the carbohydrate consumption calculated from oxygen uptake suggests that previous estimates, using carbon dioxide evolution as a measure of the cost of nitrogen fixation may be underestimates. Lag periods observed in the reduction of acetylene to ethylene suggest that there is a resistance to diffusion of gases in the root nodules. 相似文献
55.
In a comparative study the requirement of several strains of autotrophic hydrogen-oxidizing bacteria for nickel was examined. Autotrophic growth was studied both in liquid media, previously freed from trace metals; and on solidified media, using a plate diffusion assay. The latter assay was based on the observation that EDTA causes complete inhibition of autotrophic growth on agar medium as a result of nickel deficiency. Nickel was shown to be required as a trace element in five strains of Alcaligenes eutrophus, in two strains of Xanthobacter autotrophicus, in Pseudomonas flava, in Arthrobacter spec. 11X and in strain 12X. In these bacteria nickel was not replaceable by cobalt, copper, manganese or zinc ions. No significant nickel requirement was detected by these methods, however, for Paracoccus denitrificans and Nocardia opaca 1b. 相似文献
56.
(1) The for 1.3 mM D-allose uptake and efflux in insulin-stimulated adipocytes is 1.7 ± 0.1 min. In the absence of insulin mediated uptake of D-allose is virtually eliminated and the uptake rate () is near that calculated for nonmediated transport. The kinetic parameters for D-allose zero-trans uptake in insulin-treated cells are Kztoi = 271.3 ± 34.2 mM, Vztoi = 1.15 ± 0.12 mM · s?1. (2) A kinetic analysis of the single-gate transporter (carrier) model interacting with two substrates (or substrate plus inhibitor) is presented. The analysis shows that the heteroexchange rates for two substrates interacting with the transporter are not unique and can be calculated from the kinetic parameters for each sugar acting alone with the transporter. This means that the equations for substrate analogue inhibition of the transport of a low affinity substrate such as D-allose can be simplified. It is shown that for the single gate transporter the Ki for a substrate analogue inhibitor should equal the equilibrium exchange Km for this analogue. (3) Analogues substituted at C-1 show a fused pyranose ring is accepted by the transporter. 1-Deoxy-D-glucose is transported but has low affinity for the transporter. High affinity can be restored by replacing a fluorine in the β-position at C-1. The Ki for ; the Ki for . Replacing the ring oxygen also results in a marked reduction in affinity. The Ki for . (4) A hydroxyl in the gluco configuration at C-2 is not required as 2-deoxy-d-galactose (Ki = 20.75 mM) has a slightly higher affinity than d-galactose (Ki = 24.49 mM). A hydroxyl in the manno configuration at C-2 interferes with transport as d-talose (Ki = 35.4 mM) has a lower affinity than d-galactose. (5) d-Allose (Km = 271.3 mM) and 3-deoxy-d-glucose (Ki = 40.31 mM) have low affinity but high affinity is restored by substituting a fluorine in the gluco configuration at C-3. The Ki for . (6) Analogues modified at C-4 and C-6 do not show large losses in affinity. However, 6-deoxy-d-glucose (Ki = 11.08 mM) has lower affinity than d-glucose and 6-deoxy-d-galactose Ki = 33.97 mM) has lower affinity than d-galactose. Fluorine substitution at C-6 of d-galactose restores high affinity. The Ki for . Removal of the C-5 hydroxymethyl group results in a large affinity loss. The Ki. The Ki for . (7) These results indicate that the important hydrogen bonding positions involved in sugar interaction with the insulin-stimulated adipocytes transporter are the ring oxygen, C-1 and C-3. There may be a weaker hydrogen bond to C-6. Sugar hydroxyls in non-gluco configurations may sterically hinder transport. 相似文献
57.
A quantitative study was made of macromolecular (nucleic acids, protein), carbohydrate and mineral (magnesium, potassium and phosphorus) components of Aspergillus nidulans in glucose limited chemostat cultures, under varying conditions of dilution rate, temperature, pH and NaCl concentration.The overall mineral content showed greatest variation in response to changes in culture salinity, which also affected the mycelial carbohydrate content. Concomitant and opposite changes in the conent of cations and carbohydrates under conditions of increasing salinity may be interpreted in terms of mycelial osmoregulation. Slight variations in DNA content but gross fluctuations in the level of RNA were noted under the different cultural conditions examined. Co-ordinate changes in RNA and Mg2+ contents were evident only under certain conditions: dilution rate from 0.05–0.07 h-1 or temperature from 22–30° C. The constant molar stoichiometry between RNA and Mg2+ characteristic of unicellular microorganisms was not a feature of fungal growth. The protein content was most affected by shifts of temperature and reached minimal values at 25 and 50° C.The growth environment had a marked influence on the protein synthesising activity of RNA, which increased eightfold as the dilution rate was increased from 0.02–0.175 h-1, doubled within the temperature range 20–30° C and fell by 50% between 40 and 50° C. These observations are discussed in the context of the constant ribosomal efficiency in protein synthesis hypothesis. 相似文献
58.
Cytochromes c
3 of different strains of sulfatereducing bacteria have been purified and tested for their capacity to reduce colloidal sulfur to hydrogen sulfide. The results are in good agreement with the activities reported for the whole cells. Cytochrome c
3 is the sulfur reductase of some strains of sulfate-reducing bacteria such as Desulfovibrio desulfuricans Norway 4 and sulfate-reducing bacterium strain 9974 from which the sulfur reductase activity can be purified with the cytochrome c
3. In contrast, Desulfovibrio vulgaris Hildenborough cytochrome c
3 is inhibited by the product of the reaction namely hydrogen sulfide. Chloramphenicol has no effect on the sulfur reductase activity of D. desulfuricans Norway 4 when resting cells grown on lactate-sulfate medium are put in the presence of colloidal sulfur. This shows that the sulfur reductase activity is constitutive and corresponds to the fact that colloidal sulfur grown cells do not contain more cytochrome c
3 (or another sulfur reductase) than lactate-sulfate-grown cells. 相似文献
59.
When Lemna minor L. is transferred to an atmosphere with H2S, there is a rapid loss of extractable adenosine-5-phosphosulfate sulfotransferase activity. The activity is restored within 24 h in an atmosphere without H2S. This restoration of activity is completely inhibited by cycloheximid but not by chloramphenicol. In vitro, S2- up to 5 mM and cysteine, methionine, and glutathione up to 50 mM do not inhibit the enzyme. The activities of ATP sulfurylase and O-acetyl-L-serine sulfhydrylase are not affected significantly by H2S. The physiological significance of the regulation of adenosine-5-phosphosulfate sulfotransferase is discussed.Abbreviations APS
adenosine-5-phosphosulfate
- PAPS
adenosine-3-phosphate-5-phosphosulfate
- BSA
bovine serum albumin
- DTT
dithiothreitol
- POPOP
1,4-di [2-(5-phenyloxazolyl)]-benzene
- PPO
2,5-diphenyloxazol
This is no. 6 in the series Regulation of Sulfate Assmilation in Plants 相似文献
60.
Xylose anaerobic conversion by open-mixed cultures 总被引:1,自引:0,他引:1
Margarida F. Temudo Tania Mato Robbert Kleerebezem Mark C. M. van Loosdrecht 《Applied microbiology and biotechnology》2009,82(2):231-239
Xylose is, after glucose, the dominant sugar in agricultural wastes. In anaerobic environments, carbohydrates are converted
into volatile fatty acids and alcohols. These can be used as building blocks in biotechnological or chemical processes, e.g.,
to produce bioplastics. In this study, xylose fermentation by mixed microbial cultures was investigated and compared with
glucose under the same conditions. The product spectrum obtained with both substrates was comparable. It was observed that,
in the case of xylose, a higher fraction of the carbon was converted into catabolic products (butyrate, acetate, and ethanol)
and the biomass yield was approximately 20% lower than on glucose, 0.16 versus 0.21 Cmol X/Cmol S. This lower yield is likely
related to the need of an extra ATP during xylose uptake. When submitted to a pulse of glucose, the population cultivated
on xylose could instantaneously convert the glucose. No substrate preference was observed when glucose and xylose were fed
simultaneously to the continuously operated bioreactor. 相似文献