共查询到20条相似文献,搜索用时 31 毫秒
1.
The inhibition by cupric ions of the hydration of CO2 catalyzed by carbonic anhydrase II is interesting because of the results of Tuet al. obtained at chemical equilibrium, indicating that Cu2+ inhibits specifically a proton transfer in the catalytic pathway. We have measured this inhibition at steady state, using stopped-flow methods. The inhibition by Cu2+ of the hydration of CO2 catalyzed by carbonic anhydrase II had aK I near 1×10?6 M atpH 7.0 and gave inhibition that is noncompetitive atpH 6.0 and mixed, but close to uncompetitive, atpH 6.8. ThepH dependence of this binding is consistent with a binding site for Cu2+ on the enzyme with apK a near 7. The binding interaction between Cu2+ and the fluorescent inhibitor 5-dimethylaminonaphthalene-l-sulfonamide on carbonic anhydrase II was noncompetitive, indicating that the binding site for Cu2+ is distinct from the coordination sphere of zinc in which the actual interconversion of CO2 and HCO 3 ? and the binding of sulfonamides takes place. 相似文献
2.
The carbon dioxide hydration activity of brush-border carbonic anhydrase from the dog kidney 总被引:2,自引:0,他引:2
The steady-state kinetic parameters for the hydration of CO2 catalyzed by membrane-bound carbonic anhydrase from the renal brush-border of the dog are compared with the same parameters for water-soluble bovine erythrocyte carbonic anhydrase. For the membrane-bound enzyme, the turnover number kcat is 6.5 × 105 s?1 and the Michaelis constant is 7.5 mm for CO2 hydration at pH 7.4 and 25 °C. The corresponding constants for bovine carbonic anhydrase under these conditions are 6.3 × 105 s?1 and 15 mm (Y. Pocker and D.W. Bjorkquist (1977)Biochemistry16, 5698–5707). The rate constant for the transfer of a proton between carbonic anhydrase and buffer was determined from the dependence of the catalytic rate on the concentration of the buffers imidazole and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes); the value of 2 × 108m?1s?1 describes this constant for both forms of carbonic anhydrase at pH 7.4. Furthermore, the pH dependence of the initial velocity of hydration of CO2 in the range of pH 6.5 to 8.0 is identical for the membrane-bound and soluble enzyme at low buffer concentration (1–2 mm imidazole). We conclude that the membrane plays no detectable role in affecting the CO2 hydration activity and that the active site of the renal, membrane-bound carbonic anhydrase is exposed to the aqueous phase. 相似文献
3.
Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO2 hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a Km of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (Ki = 1.22 × 10?3M), arsenate (Ki = 1.17 × 10?3M), and molybdate (Ki = 1.34 × 10?7M), with these inhibitors having no effect on the CO2 hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO2 hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO2 hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites. 相似文献
4.
Investigations on the effects of Cu2+ on the structure and function of human serum albumin
下载免费PDF全文
![点击此处可从《Luminescence》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Human serum albumin (HSA) is the most prominent protein in blood plasma with important physiological functions. Although copper is an essential metal for all organisms, the massive utilization of copper has led to concerns regarding its potential health impact. To better understand the potential toxicity and toxic mechanisms of Cu2+, it is of vital importance to characterize the interaction of Cu2+ with HSA. The effect of Cu2+ on the structure and function of HSA in vitro were investigated by biophysical methods including fluorescence techniques, circular dichroism (CD), time‐resolved measurements, isothermal titration calorimetry (ITC), molecular simulations and esterase activity assay. Multi‐spectroscopic measurements proved that Cu2+ quenched the intrinsic fluorescence of HSA in a dynamic process accompanied by the formation of complex and alteration of secondary structure. But the Cu2+ had minimal effect on the backbone and secondary structure of HSA at relatively low concentrations. The ITC results indicated Cu2+ interacted with HSA spontaneously through hydrophobic forces with approximately 1 thermodynamic identical binding sites at 298 K. The esterase activity of HSA was inhibited obviously at the concentration of 8 × 10‐5 M. However, molecular simulation showed that Cu2+ mainly interacted with the amino acid residues Asp (451) by the electrostatic force. Thus, we speculated the interaction between Cu2+ and HSA might induce microenvironment of the active site (Arg 410). This study has provided a novel idea to explore the biological toxicity of Cu2+ at the molecular level. Copyright © 2015 John Wiley & Sons, Ltd. 相似文献
5.
Xican Li Hong Xie Qian Jiang Gang Wei Lishan Lin Changying Li Xingmei Ou Lichan Yang Yulu Xie Zhen Fu Yamei Liu Dongfeng Chen 《Cellular & molecular biology letters》2017,22(1):31
The natural dihydroflavonol (+) taxifolin was investigated for its protective effect on Fenton reagent-treated bone marrow-derived mesenchymal stem cells (bmMSCs). Various antioxidant assays were used to determine the possible mechanism. These included ?OH-scavenging, 2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide radical-scavenging (PTIO?-scavenging), 1, 1-diphenyl-2-picryl-hydrazl radical-scavenging (DPPH?-scavenging), 2, 2′-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) radical-scavenging (ABTS+?-scavenging), Fe3+-reducing, and Cu2+-reducing assays. The Fe2+-binding reaction was also investigated using UV-Vis spectra. The results revealed that cell viability was fully restored, even increasing to 142.9?±?9.3% after treatment with (+) taxifolin. In the antioxidant assays, (+) taxifolin was observed to efficiently scavenge ?OH, DPPH? and ABTS+? radicals, and to increase the relative Cu2+- and Fe3+-reducing levels. In the PTIO?-scavenging assay, its IC50 values varied with pH. In the Fe2+-binding reaction, (+) taxifolin was found to yield a green solution with two UV-Vis absorbance peaks: λmax =?433 nm (ε =5.2?×?102 L mol?1 cm ?1) and λmax =?721 nm (ε?=?5.1?×?102 L mol?1 cm ?1). These results indicate that (+) taxifolin can act as an effective ?OH-scavenger, protecting bmMSCs from ?OH-induced damage. Its ?OH-scavenging action consists of direct and indirect antioxidant effects. Direct antioxidation occurs via multiple pathways, including ET, PCET or HAT. Indirect antioxidation involves binding to Fe2+. 相似文献
6.
The inhibition by cupric ions of the hydration of CO2 catalyzed by carbonic anhydrase II is interesting because of the results of Tuet al. obtained at chemical equilibrium, indicating that Cu2+ inhibits specifically a proton transfer in the catalytic pathway. We have measured this inhibition at steady state, using stopped-flow methods. The inhibition by Cu2+ of the hydration of CO2 catalyzed by carbonic anhydrase II had aK
I
near 1×10–6 M atpH 7.0 and gave inhibition that is noncompetitive atpH 6.0 and mixed, but close to uncompetitive, atpH 6.8. ThepH dependence of this binding is consistent with a binding site for Cu2+ on the enzyme with apK
a
near 7. The binding interaction between Cu2+ and the fluorescent inhibitor 5-dimethylaminonaphthalene-l-sulfonamide on carbonic anhydrase II was noncompetitive, indicating that the binding site for Cu2+ is distinct from the coordination sphere of zinc in which the actual interconversion of CO2 and HCO
3
–
and the binding of sulfonamides takes place. 相似文献
7.
A 36-amino acid residue peptide containing the presumed metal-binding ligands at the active site of human erythrocyte carbonic anhydrase B was synthesized by the standard solid phase method. The synthetic peptide was purified by ion-exchange chromatography and was homogeneous as judged by cellulose acetate gel electrophoresis. Amino acid analysis, dansylation, C-terminal determination, and four cycles of Edman degradation all gave results consistent with the anticipated sequence. The peptide binds Co(II) with an apparent dissociation constant of about 7 × 10?5M (uncorrected) but has little, if any, of the catalytic activity of carbonic anhydrase. Possible explanations for the weak binding of the metal ion are discussed along with prospects and strategies for designing polypeptide models of enzymatic catalysts. 相似文献
8.
The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co2+, Cu2+, and Fe3+, increased the enzyme activity, whereas CA activity was inhibited by Pb2+, Hg2+, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO2 to CaCO3. The maximum CO2 sequestration potential was achieved with immobilized CA (480 mg CaCO3/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO2 sequestration. 相似文献
9.
D N Silverman 《Archives of biochemistry and biophysics》1973,155(2):452-457
Oxygen-18 exchange techniques were applied to the dehydration of bicarbonate catalyzed by human carbonic anhydrase C. The rates of depletion of oxygen-18 from labeled bicarbonate were measured for both the catalyzed and uncatalyzed reactions at pH 9.4 and 25 °C. The equilibrium dissociation constant of the enzyme-substrate complex K is 0.321 ± 0.040 m and is (8.3 ± 1.9) × 105m?1 sec?1 under these conditions. On the basis of these results it is demonstrated that the oxygen-18 exchange technique is capable of measuring K and kenz for the carbonic anhydrase catalyzed dehydration of bicarbonate at a high pH range in which other kinetic techniques are not effective.It was also shown that the oxygen-18 exchange technique is an effective micromethod for the determination of carbonic anhydrase. Rates of isotopic depletion of labeled bicarbonate (in solutions of the enzyme) which fall outside the limits of error for the uncatalyzed rate of depletion demonstrate that this technique can detect concentrations of human carbonic anhydrase C as low as 5 × 10?11m. 相似文献
10.
Hydrogen peroxide (H2O2) in minute quantity serves as a signalling molecule. However, the role of H2O2 in combination with brassinosteroids (stress regulators) in plants under toxic levels of copper, is poorly understood. With an aim to explore and elaborate their role in plants subjected to abiotic stress, the surface sterilized seeds of mung bean (Vigna radiata) were sown in earthen pots filled with soil and manure enriched with different levels of Cu2+ (50 or 100 mg kg?1 of soil) and allowed to grow under natural environmental conditions. At 15 and 20 days stage, the plants were sprayed with H2O2 (2.5 mM) and/or 28-homobrassinolide (HBL, 10?5 mM), respectively. At 45 days stage, the analysis of the plants revealed that the presence of copper in the soil caused a significant decrease in growth characteristics, activity of carbonic anhydrase and nitrate reductase, relative water content, chlorophyll content and the rate of photosynthesis whereas, the activity of antioxidant enzymes (catalase, peroxidase and superoxide dismutase) and the proline accumulation in leaves increased in Cu stressed plants. However, the exogenously applied HBL and/or H2O2, in the absence of Cu-stress strongly favoured the growth, photosynthetic parameters and also improved the activity of antioxidant enzymes and the proline content. Furthermore, the combined application of HBL and H2O2 to the foliage of the stressed plants neutralized the toxic impact of all copper regimes. Therefore, we are of the opinion that these chemicals somehow maintained the homeostasis of the metal in the plants that exhibit healthy growth. 相似文献
11.
Fluorescent proteins show fluorescence quenching by specific metal ions, which can be applied towards metal biosensing applications. In order to develop metal-biosensor, we performed spectroscopic analysis of the fluorescence quenching of fluorescent protein AmCyan and mOrange2 by various metal ions. The fluorescence intensity of AmCyan was reduced to 48.54% by Co2+ and 67.77% by Zn2+; Cu2+ reduced the fluorescence emission of AmCyan to 19.30% of its maximum. The fluorescence intensity of mOrange2 was quenched by only Cu2+, to 11.48% of its maximum. When analyzed by Langmuir equation, dissociation constants for AmCyan and mOrange2 were 56.10 and 21.46 µM, respectively. The Cu2+ quenching of AmCyan and mOrange2 were reversible upon treatment with the metal chelator EDTA, indicating that the metal ions were located on the protein surface. Their model structures suggest that AmCyan and mOrange2 have novel metal-binding sites. 相似文献
12.
Binding of divalent copper ions to aspartic acid residue 52 in hen egg-white lysozyme 总被引:4,自引:0,他引:4
V I Teichberg N Sharon J Moult A Smilansky A Yonath 《Journal of molecular biology》1974,87(2):357-368
Divalent copper was found to inhibit non-competitively the lysis of Micrococcus lysodeikticus cells by hen egg-white lysozyme, with an inhibition constant Ka= 3.8 × 102m?1. The association constants of Cu2+ for lysozyme and for a derivative of lysozyme in which tryptophan residue 108 was selectively modified, were measured spectrofluorimetrieally and found to be 1.8 × 102m?1 and 1.0 × 103m?1, respectively. The electron spin resonance spectrum of Cu2+ was not affected by the addition of lysozyme, whereas many new lines appeared on addition of the modified protein. This was interpreted as evidence for the binding of Cu2+ in the neighbourhood of tryptophan 108. To unequivocally establish the site of ligation of Cu2+, crystals of lysozyme soaked in Cu2+ were examined by X-ray crystallography and the results compared to those obtained from crystals of native lysozyme. Cu2+ was found to be located 2 to 3 Å from the carboxyl side-chain of aspartic acid 52, 5 Å from the carboxyl of glutamic acid 35 and about 7 Å from tryptophan 108.The addition of a saccharide inhibitor to lysozyme was found to increase the association constant of Cu2+ for lysozyme from a value of 1.8 × 102m?1 to 6.0 × 102m?1. This finding was interpreted as indicative of a change in conformation around tryptophan 108 and glutamic acid 35 induced by the interaction of saccharides with the enzyme, which affects the metal binding properties of aspartic acid 52. 相似文献
13.
The melting temperature of a natural DNA is decreased in the presence of increasing amounts of copper ions, whereas other divalent metal ions stabilize the DNA secondary structure at low ionic strength. At 1.28 × 10?4M, Cu2+ produces a decrease of Tm depending on base composition. At very low Cu2+ concentrations (0.5 Cu2+/2 DNA-P) a stabilization of the DNA conformation appears due to an interaction between Cu2+ and phosphate groups of the DNA molecule. In this case the normal trend of GC dependence of Tm exists similar to that with Na+ and Mg2+ as counterions. If copper ions are in excess, the observed destabilization is stronger for DNAs rich in guanine plus cytosine than for those rich in adenine plus thymine. A sharp decrease of Tm occurs between 0.5–0.8 Cu2+/2 DNA-P and 1.5 Cu2+/2 DNA-P. The breadth of the transition decreases at high Cu2+ concentration with further addition of copper ions. Denaturation and renaturation experiments indicate that Cu2+ ions exceeding the phosphate equivalents interact with the bases and reduce the forces of the DNA helix conformation. Evidence is presented, that the destabilization effect produced by Cu2+ is possibly due to an interaction with guanine sites of the DNA molecule. 相似文献
14.
d ‐penicillamine‐capped cadmium telluride quantum dots (DPA‐capped CdTe QDs) were synthesized as the new fluorescent semiconductor nanocrystal in aqueous solution. Fourier transmission infrared spectroscopy, X‐ray diffraction, transmission electron microscopy, ultraviolet‐visible and photoluminescence spectroscopy were used for characterization of the QDs. Based on the quenching effect of Cu2+ ions on the fluorescence intensity of DPA‐capped CdTe QDs, a new fluorometric sensor for copper(II) detection was developed that showed good linearity over the concentration range 5 × 10–9–3 × 10–6 m with the detection limit 0.4 × 10–9 m . Owing to the strong affinity of the DPA to copper(II), the sensor showed appropriate selectivity for copper(II) compared with conventional QDs. The DPA‐capped CdTe QDs was successfully applied for determination of Cu2+ concentration in river, well and tap waters with satisfactory results. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
15.
Bovine carbonic anhydrase shows an intrinsic fluorescence which results from tryptophans located in different microenvironments. It is possible to attribute the whole fluorescence to at least two types of tryptophan.This fluorescence is differently affected by the binding of different metals. In fact while Zn2+ causes an increase of the fluorescence yield, the binding of Co2+, Cu2+ and Hg2+ is followed by a quenching of the fluorescence. The quenching is about 40% for the cobalt, 80% for the copper and 60% for the mercury derivative. The binding of Cu2+ and Hg2+ induces also a change in the shape of the fluorescence emission spectrum. This fact suggests a different influence of the metals on the various types of tryptophan.The fluorescence quenching induced by iodide which can bind to the metal and act as a fluorescence perturbing agent is also indicative of the presence of different tryptophans. 相似文献
16.
Rob van Hille Marijke Fagan Lucinda Bromfield Robert Pott 《Journal of applied phycology》2014,26(1):377-385
Threat of global warming due to carbon dioxide (CO2) emissions has stimulated research into carbon sequestration and emissions reduction technologies. Alkaline scrubbing allows CO2 to be captured as bicarbonate, which can be photochemically fixed by microalgae. The carbon concentrating mechanism (CCM), of which external carbonic anhydrase is a key component, allows organisms to utilise this bicarbonate. In order to select a suitable strain for this application, a screening tool is required. The current method for determining carbonic anhydrase activity, the Wilbur and Anderson assay, was found to be unsuitable as a screening tool as the associated error was unacceptably large and tests on whole cells were inconclusive. This paper presents the development of a new, whole cell assay to measure inorganic carbon uptake and external carbonic anhydrase activity, based on classical pH drift experiments. Spirulina platensis was successfully used to develop a correlation between the specific carbon uptake (C) and the specific pH change (dpH). The relationship is described by the following: C[mmol C (g dry algae)?1?h?1]?=?0.064?×?(dpH). Inhibitor and salt dissociation tests validated the activity and presence of external carbonic anhydrase and allowed correlation between the Wilbur and Anderson assay and the new whole cell assay. Screening tests were conducted on S. platensis, Scenedesmus sp., Chlorella vulgaris and Dunaliella salina that were found to have carbon uptake rates of 5.76, 5.86, 3.86 and 2.15 mmol C (g dry algae)?1?h?1, respectively. These results corresponded to the species' known bicarbonate utilisation abilities and validated the use of the assay as a screening tool. 相似文献
17.
Tamiika K. Hurst Da Wang Richard B. Thompson Carol A. Fierke 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(2):393-403
Carbonic anhydrases are archetypical zinc metalloenzymes and as such, they have been developed as the recognition element of a family of fluorescent indicators (sensors) to detect metal ions, particularly Zn2+ and Cu2+. Subtle modification of the structure of human carbonic anhydrase II isozyme (CAII) alters the selectivity, sensitivity, and response time for these sensors. Sensors using CAII variants coupled with zinc-dependent fluorescent ligands demonstrate picomolar sensitivity, unmatched selectivity, ratiometric fluorescence signal, and near diffusion-controlled response times. Recently, these sensors have been applied to measuring the readily exchangeable concentrations of zinc in the cytosol and nucleus of mammalian tissue culture cells and concentrations of free Cu2+ in seawater. 相似文献
18.
Microbial carbonic anhydrase promotes carbonate deposition, which is important in the formation and evolution of global carbon cycle and geological processes. A kind of bacteria producing extracellular carbonic anhydrase was selected to study the effects of temperature, pH value and Ca2+ concentration on bacterial growth, carbonic anhydrase activity and calcification rate in this paper. The results showed that the activity of carbonic anhydrase at 30 °C was the highest, which was beneficial to the calcification reaction, calcification rate of CaCO3 was the fastest in alkaline environment with the initial pH value of 9.0. When the Ca2+ concentration was 60 mM, compared with other Ca2+ concentration, CA bacteria could grow and reproduce best, and the activity of bacteria was the highest, too low Ca2+ concentration would affect the generation of CaCO3, while too high Ca2+ concentration would seriously affect the growth of bacteria and reduce the calcification rate. Finally, the mechanism of CaCO3 precipitation induced by microbial carbonic anhydrase was studied. Carbonic anhydrase can accelerate the hydration of CO2 into HCO3−, and react with OH− and Ca2+ to form CaCO3 precipitation in alkaline environment and in the presence of calcium source. 相似文献
19.
An electrochemiluminescence (ECL) sensor based on reduced graphene oxide–CdTe quantum dots (RGO–CdTe QDs) composites for detecting copper ion (Cu2+) was proposed. The ECL behaviours of the RGO–CdTe QD modified electrode were investigated with H2O2 as the co‐reactant. Quantitative detection of Cu2+ was realized as Cu2+ could effectively quench the ECL signal of the RGO–CdTe QDs. A wide linear range of 1.00 × 10?14 to 1.00 × 10?4 M (R = 0.9953) was obtained under optimized conditions, and a detection limit (S/N = 3) was achieved of as low as 3.33 × 10?15 M. The proposed sensor also exhibited good stability and selectivity for the detection of copper ions. Finally, the analytical application of the proposed sensor was also evaluated using river water. 相似文献
20.
PHILLIP E. RYALS HARRIETT E. SMITH-SOMERVILLE 《The Journal of eukaryotic microbiology》1986,33(3):382-387
ABSTRACT. Periodically, stocks of Tetrahymena vorax, which normally yield 70–90% macrostomes when subjected to heat shock or other induction methods, become low-transformers and yield ≥30% macrostomes. The addition to the post-heat-shock wash buffer (pH 6.8) of 2.7 × 10-4 M Fe3+, 1.6 × 10-5 M Cu2+, 1 × 10-4 M retinol palmitate or the adjustment of the buffer to a pH of 4 to 5 boosts transformation significantly over controls in inorganic medium alone. The addition of Fe2+ or Cu1+ has a similar, but less pronounced effect on transformation. Ferric ion (2.7 × 10-4 M) will significantly increase transformation in starved non-heat-shocked cells, whereas Fe2+, copper, retinol palmitate, and hydrogen ion concentration have no effect. The agents that boost transformation appear to act by delaying cell division in pre-transformants. Membrane fluidity, as inferred by fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene, is altered in a consistent manner by starvation and heat shock. Enhancing agents, including compounds previously shown to boost heat-shock-induced macrostome formation, produce diverse shifts in membrane fluidity. Their effect on transformation of these low-transforming cells therefore appears to be attributable to some mechanism or mechanisms other than a direct alteration of membrane physical properties. 相似文献