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1.
Kinetic parameters of photoinduced permeability increase of artificial lipid membranes, modified by ROS fragments (tau20 degrees C = 20 mesec Ea = 33 +/- 2 kcal/mole) coincides with appropriate parameters of photoinduced protein fluorescence intensity decrease and ROS fragments absorption spectra change (metarhodopsin I leads leads to metarhodopsin II transition). Hydroxylamine accelerates this process, its rate is proportional to hydroxylamine at concentrations lower than 0.6 M. 相似文献
2.
Following flash excitation, oxygen pulses and fluorescence kinetics in the time range 0-16 mus were studied in the alga Chlorella pyrenoidosa during incubation with various concentrations of hydroxylamine. The obtained results could be explained considering four effects of hydroxylamine. 1. Hydroxylamine removes (reduces) oxidizing equivalents, generated in the water-splitting system by flash excitation. This process does not markedly affect the fluorescence yield kinetics between 0 and 16 mus following the ignition of a flash and reaches a constant rate within a few minutes, but possibly within a few seconds, after addition of hydroxylamine. In a sequence of flashes separated by dark time td, the steady-state oxygen yield in the flashes is exp(-ktd), the yield at td=0 being taken equal to 1, where k=(0.1 + beta[NH2OH])s-1, with [NH2OH] in mM and beta=0.6 mM-1, provided [NH2OH]greater than or equal to 0.5 mM. 2. An inhibition between Z, the physiological donor and the oxidized reaction center pigment P+ occurs, proceeding as exp (-kiti)where ti is the incubation time with hydroxylamine and ki=(alpha[NH2OH]) min-1, with [NH2OH] in mM and alpha=0.14 mM-1. This process not only inhibits oxygen evolution capability, but also decreases the amplitude of the fluorescence yield difference deltaphi=phi(16 mus)-phi(2 mus) induced by a flesh in the steady state. In a fraction of the reaction centers this inhibition occurs "immediately" after the addition of hydroxylamine. These observations, combined with the conslusion of Cheniae and Martin (1971, Plant Physiol. 47, 568-575) that the inhibition of the Hill reaction is related to the extraction of bound manganese indicate that the reaction between Z and P+ requires bound manganese. 3. In the inhibited centers a second donor for P+, D, connected to an entry site for the artificial electron donor hydroxylamine becomes apparent. 4. A flash-induced oxygen uptake signal was observed in the presence of hydroxylamine, which was shown to be caused by a system II reaction. The effects under (1) and (4) were reversed in the dark if hydroxylamine was removed by washing. The effects under (2) and (3) were reversed during illumination of a washed sample. 相似文献
3.
D A Bazylinski R A Arkowitz T C Hollocher 《Archives of biochemistry and biophysics》1987,259(2):520-526
The reaction between hydroxylamine (NH2OH) and human hemoglobin (Hb) at pH 6-8 and the reaction between NH2OH and methemoglobin (Hb+) chiefly at pH 7 were studied under anaerobic conditions at 25 degrees C. In presence of cyanide, which was used to trap Hb+, Hb was oxidized by NH2OH to methemoglobin cyanide with production of about 0.5 mol NH+4/mol of heme oxidized at pH 7. The conversion of Hb to Hb+ was first order in [Hb] (or nearly so) but the pseudo-first-order rate constant was not strictly proportional to [NH2OH]. Thus, the apparent second-order rate constant at pH 7 decreased from about 30 M-1 X s-1 to a limiting value of 11.3 M-1 X s-1 with increasing [NH2OH]. The rate of Hb oxidation was not much affected by cyanide, whereas there was no reaction between NH2OH and carbonmonoxyhemoglobin (HbCO). The pseudo-first-order rate constant for Hb oxidation at 500 microM NH2OH increased from about 0.008 s-1 at pH 6 to 0.02 s-1 at pH 8. The oxidation of Hb by NH2OH terminated prematurely at 75-90% completion at pH 7 and at 30-35% completion at pH 8. Data on the premature termination of reaction fit the titration curve for a group with pK = 7.5-7.7. NH2OH was decomposed by Hb+ to N2, NH+4, and a small amount of N2O in what appears to be a dismutation reaction. Nitrite and hydrazine were not detected, and N2 and NH+4 were produced in nearly equimolar amounts. The dismutation reaction was first order in [Hb+] and [NH2OH] only at low concentrations of reactants and was cleanly inhibited by cyanide. The spectrum of Hb+ remained unchanged during the reaction, except for the gradual formation of some choleglobin-like (green) pigment, whereas in the presence of CO, HbCO was formed. Kinetics are consistent with the view advanced previously by J. S. Colter and J. H. Quastel [1950) Arch. Biochem. 27, 368-389) that the decomposition of NH2OH proceeds by a mechanism involving a Hb/Hb+ cycle (reactions [1] and [2]) in which Hb is oxidized to Hb+ by NH2OH. 相似文献
4.
Self-association of the polyene antibiotic nystatin in dipalmitoylphosphatidylcholine vesicles: a time-resolved fluorescence study.
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The interaction between Nystatin and small unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, both in gel (T = 21 degrees C) and in liquid-crystalline (T = 45 degrees C) phases, was studied by steady-state and time-resolved fluorescence measurements by taking advantage of the intrinsic tetraene fluorophore present in this antibiotic. It was shown that Nystatin aggregates in aqueous solution with a critical concentration of 3 microM. The enhancement in the fluorescence intensity of the antibiotic was applied to study the membrane binding of Nystatin, and it was shown that the antibiotic had an almost fivefold higher partition coefficient for the vesicles in a gel (P = (1.4 +/- 0.1) x 10(3)) than in a liquid-crystalline phase (P = (2.9 +/- 0.1) x 10(2)). Moreover, a time-resolved fluorescence study was used to examine Nystatin aggregation in the membrane. The emission decay kinetics of Nystatin was described by three and two exponentials in the lipid membrane at 21 degrees C and 45 degrees C, respectively. Nystatin mean fluorescence lifetime is concentration-dependent in gel phase lipids, increasing steeply from 11 to 33 ns at an antibiotic concentration of 5-6 microM, but the fluorescence decay parameters of Nystatin were unvarying with the antibiotic concentration in fluid lipids. These results provide evidence for the formation of strongly fluorescent antibiotic aggregates in gel-phase membrane, an interpretation that is at variance with a previous study. However, no antibiotic self-association was detected in a liquid-crystalline lipid bilayer within the antibiotic concentration range studied (0-14 microM). 相似文献
5.
High-Temperature Induced Chlorophyll Fluorescence Rise in Plants at 40–50 °C: Experimental and Theoretical Approach 总被引:1,自引:0,他引:1
We studied the temperature dependence of chlorophyll fluorescence intensity in barley leaves under weak and actinic light excitation during linear heating from room temperature to 50 degrees C. The heat-induced fluorescence rise usually appearing at around 40-50 degrees C under weak light excitation was also found in leaves treated with 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) or hydroxylamine (NH(2)OH). However, simultaneous treatment with both these compounds caused a disappearance of the fluorescence rise. We have suggested that the mechanism of the heat-induced fluorescence rise in DCMU-treated leaves is different than that in untreated or NH(2)OH-treated leaves. In DCMU-treated leaves, the heat-induced fluorescence rise reflects an accumulation of Q(A) (-) even under weak light excitation due to the thermal inhibition of the S(2)Q(A) (-) recombination as was further documented by a decrease in the intensity of the thermoluminescence Q band. Mathematical model simulating this experimental data also supports our interpretation. In the case of DCMU-untreated leaves, our model simulations suggest that the heat-induced fluorescence rise is caused by both the light-induced reduction of Q(A) and enhanced back electron transfer from Q(B) to Q(A). The simulations also revealed the importance of other processes occurring during the heat-induced fluorescence rise, which are discussed with respect to experimental data. 相似文献
6.
Lu X Huang WH Ai F Wang ZL Cheng JK 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,857(2):347-351
A method of indirectly measuring pyruvic acid (PA) by capillary electrophoresis with amperometric detection is proposed for the first time. It is based on the oximation reaction between PA and hydroxylamine (NH(2)OH), and the quantification of PA was performed by direct and sensitive amperometric detection of excessive NH(2)OH after the oximation reaction. This method displayed a good sensitivity, and the detection limits of NH(2)OH and PA are 1.76 x 10(-7) and 3.88 x 10(-7)mol/L, respectively at S/N=3. The linear relationship between the peak current and PA concentration is exhibited over the range from 4 x 10(-6) to 1 x 10(-4)mol/L. This method has been applied to determine PA in rat plasma with satisfactory results. 相似文献
7.
Linkage isomerization reaction of intrastrand cross-links in trans-diamminedichloroplatinum(II)-modified single-stranded oligonucleotides. 总被引:2,自引:2,他引:0
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The stability of trans-(Pt(NH3)2[d(CGAG)-N7-G,N7-G]) adducts, resulting from cross-links between two guanine residues at d(CGAG) sites within single-stranded oligonucleotides by trans-diamminedichloro-platinum(II), has been studied under various conditions of temperature, salt and pH. The trans-(Pt(NH3)2[d(C GAG)-N7-G,N7-G]) cross-links rearrange into trans-(Pt(NH3)2[d(CGAG)-N3-C,N7-G]) cross-links. The rate of rearrangement is independent of pH, in the range 5-9, and of the nature and concentration of the salt (NaCl or NaCIO4) in the range 10-400 mM. The reaction rate depends upon temperature, the t1/2 values for the disappearance of the (G,G) intrastrand cross-link ranging from 120 h at 30 degrees C to 70 min at 80 degrees C. The linkage isomerization reaction occurs in oligonucleotides as short as the platinated tetramer d(CGAG). Replacement of the intervening residue A by T has no major effect on the reaction. The C residue adjacent to the adduct on the 5' side plays a key-role in the reaction; its replacement by a G, A or T residue prevents the reaction occuring. No rearrangement was observed with the C residue adjacent to the adduct on the 3' side. It is proposed that the linkage isomerization reaction results from a direct attack of the base residue on the platinum(II) square complex. 相似文献
8.
Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O----HNO2 + 2e- + 2 H+. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants k1 and k2. CO binds to ferrous P460 with apparent first-order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15 degrees C to 20.7 degrees C and at hydrostatic pressures ranging over 0.1-80 MPa. From the measurements, thermodynamic parameters delta V+ (activation volume), delta G+, delta H+, and delta S+ have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on delta V+ (-30 ml.mol-1), delta G+, delta H+ or delta S+ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between ln k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml.mol-1 to 57 ml.mol-1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0 degrees C and, in water or ethylene glycol, the linear dependence of ln k1 on P was displaced downwards as the temperature changed from 3.5 degrees C to -15 degrees C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml.mol-1 in ethylene glycol. The activation volume increased with increasing temperature.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
9.
Clinical and veterinary uses of growth hormone-releasing factor [GRF(1- 29)NH2] require the design of analogs that are resistant to proteolysis by serum and liver degrading enzymes. This study investigated rat GRF(1-29)NH2 processing in serum and liver homogenate by means of high pressure liquid chromatography (HPLC). Synthetic rGRF(1-29)NH2 (30 microM) was incubated (0-120 min, 37 degrees C) in serum (49 +/- 8 mg prot./ml). The rGRF(1-29)NH2 (10 microM) was also incubated (0-120 min, 37 degrees C) with liver homogenate (200 +/- 6 micrograms prot./ml). Time course studies of rGRF(1-29)NH2 disappearance showed apparent half-lives of 18 +/- 4 min and 13 +/- 3 min in serum and liver homogenate, respectively. This was accompanied by the appearance of degradation products that were all less hydrophobic than the native peptide. In the serum, two major metabolites were detected and isolated by preparative HPLC. Combined results of amino acid analysis, sequencing, and chromatography with synthetic homologs revealed the presence of rGRF(1-20)OH and (3-20)OH. A small amount of rGRF(12-29)NH2, coeluting with rGRF(3-20)OH, was also found by sequencing. In the liver, rGRF(1-18)OH, (3-18)OH, and (1-10)OH were identified. The peptide bond Ala2-Asp3 (DPP IV cleavage site) was hydrolyzed in both serum and liver. Other tissue-specific cleavage sites were Arg11-Arg12 and Arg20-Lys21 (trypsin-like cleavage site) in the serum, and Tyr10-Arg11 and Tyr18-Ala19 (chymotrypsin-like cleavage site) in the liver.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
M P Jackman M A Parry J Hofsteenge S R Stone 《The Journal of biological chemistry》1992,267(22):15375-15383
Stopped-flow fluorescence spectroscopy has been used to study the reaction of human alpha-thrombin with recombinant hirudin variant 1 (rhir) at 37 degrees C and an ionic strength of 0.125 M. A 35% enhancement in intrinsic fluorescence accompanied formation of the thrombin-rhir complex. Over one third of this enhancement corresponded to a structural change that could be induced by binding of either the NH2-terminal fragment (residues 1-51) or the COOH-terminal fragment (residues 52-65) of rhir. Three kinetic steps were detected for reaction of thrombin with rhir. At high rhir concentrations (greater than or equal to 3 microM), two intramolecular steps with observed rate constants of 296 +/- 5 s-1 and 50 +/- 1 s-1 were observed. By using the COOH-terminal fragment of rhir as a competitive inhibitor, it was possible to obtain an estimate of 2.9 x 10(8) M-1 s-1 for the effective association rate constant at low rhir concentrations. At higher ionic strengths, this rate constant was lower, which is consistent with the formation of the initial complex involving an ionic interaction. The mechanism for the reaction of both the COOH- and NH2-terminal fragments of rhir appeared to involve two steps. When thrombin was reacted with the COOH-terminal fragment at high concentrations (greater than or equal to 6 microM), the bimolecular step occurred within the dead time of the spectrometer and only one intramolecular step, with a rate constant of 308 +/- 5 s-1 was observed. At concentrations of NH2-terminal fragment below 50 microM, its binding to thrombin appeared to be a bimolecular reaction with an association rate constant of 8.3 x 10(5) M-1 s-1. In the presence of saturating concentrations of the COOH-terminal fragment, a 1.7-fold increase in this rate constant was observed. At concentrations of NH2-terminal fragment greater than 50 microM, biphasic reaction traces were observed which suggests a two-step mechanism. By comparing the reaction amplitudes and dissociation constants observed with rhir and its COOH-terminal fragment, it was possible to obtain approximate estimates for the values of the rate constants of different steps in the formation of the rhir-thrombin complex. 相似文献
11.
Cabello P Pino C Olmo-Mira MF Castillo F Roldán MD Moreno-Vivián C 《The Journal of biological chemistry》2004,279(44):45485-45494
Rhodobacter capsulatus E1F1 grows phototrophically with nitrate as nitrogen source. Using primers designed for conserved motifs in bacterial assimilatory nitrate reductases, a 450-bp DNA was amplified by PCR and used for the screening of a genomic library. A cosmid carrying an insert with four SalI fragments of 2.8, 4.1, 4.5, and 5.8 kb was isolated, and DNA sequencing revealed that it contains a nitrate assimilation (nas) gene region, including the hcp gene coding for a hybrid cluster protein (HCP). Expression of hcp is probably regulated by a nitrite-sensitive repressor encoded by the adjacent nsrR gene. A His(6)-HCP was overproduced in Escherichia coli and purified. HCP contained about 6 iron and 4 labile sulfide atoms per molecule, in agreement with the presence of both [2Fe-2S] and [4Fe-2S-2O] clusters, and showed hydroxylamine reductase activity, forming ammonia in vitro with methyl viologen as reductant. The apparent K(m) values for NH(2)OH and methyl viologen were 1 mM and 7 microM, respectively, at the pH and temperature optima (9.3 and 40 degrees C). The activity was oxygen-sensitive and was inhibited by sulfide and iron reagents. R. capsulatus E1F1 grew phototrophically, but not heterotrophically, with 1 mM NH(2)OH as nitrogen source, and up to 10 mM NH(2)OH was taken up by anaerobic resting cells. Ammonium was transiently accumulated in the media, and its assimilation was prevented by L-methionine-D,L-sulfoximine, a glutamine synthetase inhibitor. In addition, hydroxylamine- or nitrite-grown cells showed the higher hydroxylamine reductase activities. However, R. capsulatus B10S, a strain lacking the whole hcp-nas region, did not grow with 1 mM NH(2)OH. Also, E. coli cells overproducing HCP tolerate hydroxyl-amine better during anaerobic growth. These results suggest that HCP is involved in assimilation of NH(2)OH, a toxic product that could be formed during nitrate assimilation, probably in the nitrite reduction step. 相似文献
12.
Microwave induced transitions in zero magnetic field have been observed in the photoinduced triplet of chloroplasts treated with dithionite by monitoring changes in the intensity of the 735 nm fluorescence band at 2 degrees K. Similar results were obtained with chloroplasts treated with hydroxylamine plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The zero field parameters are D = 0.02794 +/- 0.00007 cm-1, E = 0.00382 +/- 0.00007 cm-1, i.e. equal to those of monomeric chlorophyll a to within the experimental error. The photoinduced triplet appears to be linked to Photosystem II. This indicates that the low temperatures 735 nm fluorescence band of chloroplasts is at least partly due to Photosystem II. 相似文献
13.
The kinetics of fluorescence yield inChlorella pyrenoidosa and spinach chloroplasts were studied in the time range of 0.5 μs to several hundreds of microseconds in the presence of hydroxylamine. Fluorescence was excited with a just-saturating xenon flash with a halfwidth of 13 μs (λ = 420 nm). The fast rise of the fluorescence yield which was limited by the rate of light influx, was, in the presence of 10−3–10−2 M hydroxylamine, replaced by a slow component which had a half risetime of 25 μs in essence independent of light intensity. This slow fluorescence yield increase reflects a dark reaction on the watersplitting side of Photosystem II. Simultaneous oxygen evolution measurements suggested that a fast fluorescence component is only present in organisms with intact O2-evolving system, whereas a slow rise predominantly occurs in organisms with the watersplitting system irreversibly inhibited by hydroxylamine.
The results can be explained by the following hypotheses: (a) The primary donor of Photosystem II in its oxidized state, P+, is a fluorescence quencher. (b) Hydroxylamine prevents the secondary electron donor Z from reducing the oxidized reaction center pigment P+ rapidly. This inhibition is dependent on hydroxylamine concentration and is complete at a concentration of 10−2 M. (c) A second donor (not transporting electrons from water) transfers electrons to P+ with a half time of roughly 25 μs. 相似文献
14.
The effect of hydroxylamine on the activity and aggregate structure of autotrophic nitrifying bioreactor cultures 总被引:1,自引:0,他引:1
Harper WF Terada A Poly F Le Roux X Kristensen K Mazher M Smets BF 《Biotechnology and bioengineering》2009,102(3):714-724
Addition of hydroxylamine (NH2OH) to autotrophic biomass in nitrifying bioreactors affected the activity, physical structure, and microbial ecology of nitrifying aggregates. When NH2OH is added to nitrifying cultures in 6-h batch experiments, the initial NH3-N uptake rates were physiologically accelerated by a factor of 1.4-13. NH2OH addition caused a 20-40% decrease in the median aggregate size, broadened the shape of the aggregate size distribution by up to 230%, and caused some of the microcolonies to appear slightly more dispersed. Longer term NH2OH addition in fed batch bioreactors decreased the median aggregate size, broadened the aggregate size distribution, and decreased NH3-N removal from >90% to values ranging between 75% and 17%. This altered performance is explained by quantitative fluorescence in situ hybridization (FISH) results that show inhibition of nitrifying populations, and by qPCR results showing that the copy numbers of amoA and nxrA genes gradually decreased by up to an order-of-magnitude. Longer term NH2OH addition damaged the active biomass. This research clarifies the effect of NH2OH on nitrification and demonstrates the need to incorporate NH2OH-related dynamics of the nitrifying biomass into mathematical models, accounting for both ecophysiological and structural responses. 相似文献
15.
The phosphoryl group on the serine residue at the active site of phosphoglucomutase is presumed to undergo nucleophilic attack by the monophosphate substrates glucose 1- and glucose 6-phosphate to form glucose 1,6-diphosphate. Fluoride, hydroxylamine, and several thiol compounds have now been shown to serve as effective nucleophiles toward the active phosphate and result in the dephosphorylation of phosphoglucomutase. The more extensively studied nucleophiles, cysteine, hydroxylamine, and fluoride, are effective at a concentration as low as 1 mM with a relative reactivity of 40, 2, and 1, respectively. The reaction proceeds as long as the catalytic activity of the enzyme is maintained. Inactivation of the enzyme abolishes dephosphorylation by all nucleophilic reagents thus far studied. The dephosphorylation reaction shows optimal activity of pH 6.5. The rate of dephosphorylation exhibits saturation kinetics. With fluoride the Km is 534 mM. Dephosphorylation by fluoride is stimulated by some but not all bivalent cations. Cu+ and Co2+ are the most effective. Cu2+ not only augments the reaction with fluoride but also facilitates a nucleophilic attack by water, in the absence of the halogen, to yield inorganic phosphate. No augmentation of the rate of dephosphorylation by bivalent cations can be elicited with either cysteine or hydroxylamine. The products of the fluoride reaction are phosphorofluoridate, a small but variable amount of inorganic phosphate, and a fully active dephosphoenzyme. By constrast, cysteine and hydroxylamine yield inorganic phosphate and a partially inactive enzyme. The dephosphorylation rate varies with temperature. Arrhenius plots for the fluoride reaction reveal two distinct slopes. The heat of activation between 5-37 degrees was found to be 10.2 Cal per mol. Between 0-5 degrees, however, it was considerably greater amounting to 24.3 Cal per mol. 相似文献
16.
Methane hydroxylase (MH) from the obligate methane assimilating culture of Methylococcus thermophilus catalyses oxygenation of both CH4+ and NH4+; therefore, we studied the specificity of enzyme systems catalysing the subsequent oxidation of compounds produced upon the oxygenation of these substrates (CH3OH and NH2OH). CH3OH and NH2OH were shown to be oxidized by different enzymes, viz. methanol dehydrogenase (MD) and hydroxylamine oxidase (HO), respectively. Similar to MH, MD is characterized by the absence of strict substrate specificity, and catalyses oxidation of primary alcohols other than methanol, rather than hydroxylamine. HO catalyses oxidation of hydroxylamine rather than methanol and possesses the activity of hydroxylamine:cytochrome c oxidoreductase. The constitutive character of HO from the methane assimilating bacteria and the substrate specificity of the enzyme suggest that a lithotrophic pathway for producing energy operates in these bacteria. The HO of Methylococcus thermophilus is similar in certain properties to the HO of the nitrifying bacterium Nitrosomonas europaea. 相似文献
17.
Sulfide inhibition of photosystem II in cyanobacteria (blue-green algae) and tobacco chloroplasts. 总被引:1,自引:0,他引:1
The present study shows that in the presence of 600 nm light, sulfide acts as a specific inhibitor of photosynthetic electron transport between water and Photosystem II in the cyanobacteria Aphanothece halophytica and Synechococcus 6311 as well as in tobacco chloroplasts. In the presence of 600 nm light sulfied affects the fast fluorescence transients as does a low concentration (10 mM) of hydroxylamine; the fluorescence yield decreases in the presence of either chemical and can be restored by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. In chloroplasts, however, NH2OH, an electron donor at high concentrations (40 mM), relieves the sulfide effect. In the dark, sulfide affects the cyanobacterial fluorescence transients through decrease of oxygen tension. The fluorescence yield increases in a similar pattern to that observed under nitrogen flushing. Upon omission of sulfide in A. halophytica, the characteristic aerobic fluorescence transients return, consistent with the ease of alternation between oxygenic and sulfide-dependent anoxygenic photosynthesis in many cyanobacteria. 相似文献
18.
The kinetics of water exchange across the membrane of class II chloroplasts has been studied by two NMR methods. Both methods utilize Dy(en)3+ (en = ethylenediamine) to induce a transmembranal chemical shift the order of 40 Hz in the water proton resonance. The shift reagent is impermeant to the chloroplast membrane, inert as a redox reagent, soluble at millimolar concentrations at neutral pH, and associated with a large, virtually temperature independent molar shift (0.10-0.12 ppm/mM). Water exchange across the membrane is monitored by two independent experiments. In the first, chemical exchange causes line broadening in the water proton resonance in the high-resolution spectrum. Measurement of the incremental linewidth as a function of transmembranal chemical shift determines the exchange kinetics as well as the fractions of water protons in internal and external media. In the second experiment, chemical exchange causes the transverse relaxation time, as measured by the Carr-Purcell-Gill-Meiboom technique, to be dependent on the 180 degree pulse spacing. The two experiments, while independent of each other, depend on the same set of theoretical parameters. These parameters are overdetermined by simultaneous analysis of both experiments. The mean lifetime of a water proton in the inner thylakoid space is found to be 1.1 +/- 0.8 ms at 25 degrees C and 2.75 +/- 0.4 ms at 3 degrees C in NH2OH/EDTA-treated chloroplasts. Values derived from dark-adapted chloroplasts that are active with respect to oxygen evolution are 1.1 +/- 0.3 ms (25 degrees C) and 1.75 +/- 0.4 ms (3 degrees C). The internal thylakoid volume is also determined in principle by the data, but uncertainties in the membrane volume and the transmembranal chemical shift severely limits the accuracy of this measurement. 相似文献
19.
During oxidation of hydroxylamine, hydroxylamine oxidoreductase (HAO) transfers two electrons to tetraheme cytochrome c554 at rates sufficient to account for physiological rates of oxidation of ammonia to nitrite in Nitrosomonas europaea. Spectroscopic changes indicate that the two electrons are taken up by a high-potential pair of hemes (E degrees' = +47 mV) (one apparently high spin and one low spin). During single-turnover experiments, in which the reduction of oxidized cytochrome c554 by NH2OH-reduced HAO is monitored, one electron is taken up by the high-spin heme at a rate too fast to monitor directly (greater than 100 s-1) but which is inferred either by a loss of amplitude (relative to that observed under multiple-turnover conditions) or is slowed down by increasing ionic strength (greater than or equal to 300 mM KCl). The second electron is taken up by the low-spin heme at a 10-30-fold slower rate. The latter kinetics appear multiphasic and may be complicated by a transient oxidation of HAO due to the rapid transfer of the first electron into the high-spin heme of cytochrome c554. Under multiple-turnover conditions, a "slower" rate of reduction is observed for the high-spin heme of cytochrome c554 with a maximum rate constant of approximately 30 s-1, a value also obtained for the reduction, by NH2OH, of the cytochrome c554 high-spin heme within an oxidized HAO/c554 complex. Under these conditions, the maximum rate of reduction of the low-spin heme was approximately 11.0 s-1. Both rates decreased as the concentration of cytochrome c554 was increased above the concentration of HAO.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
Re-activation of the peptidyltransferase centre of rabbit reticulocyte ribosomes after inactivation by exposure to low concentrations of magnesium ion.
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1. The larger subrivosomal particles of rabbit reticulocytes retained full activity in the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis after 4h at 0 degrees C when buffered 0.5M-NH4Cl/10-30mM-MgCl2 was the solvent. 2. Activity in the puromycin reaction was diminished to approx 10% after 15-30 min at 0 degrees C when the concentration of MgCl2 was lowered to 2mM. 3. Activity was not restored when the concentration of MgCl2 was raised from 2mM to 10-30 mM at 0 degrees C. However, activity was recovered as measured by both assay systems when the ribosome fraction was heated to 37 degrees C at the higher concentrations of MgCl2. 4. Recovery of activity was noted during the course of the polyphenylalanine synthesis in 50 mM-KCl/5mM-MgCl2/25mM-Tris/HCl, pH 7.6, at 37 degrees C. Re-activation was slow at 20 degrees C and below. 5. No more than about 5% of the protein moiety of the subparticle was lost in 0.5M-NH4Cl on decreasing MgCl2 concentration from 10mM to 2mM. No proteins were detected in the supernatant fractions by gel electrophoresis after ribosomes were separated by differential centrifugation. The supernatant fraction was not essential for the recovery of activity. However, at higher (e.g. 1M) concentrations of NH4Cl, proteins were split from the subparticle. 6. The loss and regain of activity found on lowering and restoring the concentration of MgCl2 at 0.5M-NH4Cl appears to arise from a conformational change that does not seem to be associated with a loss and regain of particular proteins. 7. A 2% decrease in E260 was noticed when the concentration of Mg2+ was restored, and the change in the spectrum indicated a net increase of approx. 100A-U base-pairs per subribosomal particle. 8. When the concentration of Mg2+ was restored, S20,W of the subparticle remained at 52+/- 1S until the sample was incubated at 37 degrees C when S20,W increased to 56 +/- 1S compared with the value of 58 +/- 1S for the subparticle as originally isolated. 相似文献