首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 968 毫秒
1.
The two Type 1 (blue) copper-binding sites of caeruloplasmin were spectroscopically differentiated by the kinetic analysis of the e.p.r. spectra during the redox cycle. One blue copper, with a hyperfine splitting constant (A parallel) of 6.8 mT, which was rapidly reduced, was not reoxidized by oxygen, whereas it was reoxidized by H2O2. The other blue copper (A parallel = 5.8 mT), which was reduced slowly, was rapidly reoxidized by either oxygen or H2O2. A conformational change of the Type 2 copper was concomitant with the fast reduction of Type 1 copper, whereas its reduction occurred during the slow phase. This sequence of events was reversed in the reoxidation step, that is, the Type 2 copper reappeared rapidly as the species with altered conformation and reverted to the symmetry typical of the native state in the slow phase. The specific reaction of a blue-copper site with the H2O2 can tentatively be related to the established ability of caeruloplasmin to prevent 'oxidative' attack of proteins and lipids.  相似文献   

2.
A study on the transfer of copper from Cu-thionein into apo-caeruloplasmin, using Cu-thionein that was previously oxidised by activated leucocytes, was performed. Cu(I)-thiolate oxidation was conveniently monitored by the progressive decline of the specific Cotton bands between 400 and 300 nm. The characteristic e.p.r. properties and NN-dimethyl-p-phenylenediamine oxidase activity indicated a successful formation of caeruloplasmin. Taking into account the simultaneous occurrence of leucocytes, apo-caeruloplasmin and Cu-thionein in blood plasma, such an interaction would favour a possible metabolic link between either copper protein.  相似文献   

3.
Hormonally produced changes in the synthesis and secretion of the serum copper-containing protein caeruloplasmin were studied in primary cultures of rat liver parenchymal cells isolated by the collagenase-perfusion technique. A rabbit antibody directed against rat caeruloplasmin was used to immunoprecipitate labelled caeruloplasmin. Isolated liver cells synthesized and secreted caeruloplasmin over a period of 3 days. Synthesis and secretion of this protein was enhanced when cells were treated with dexamethasone. The accumulation of copper was also moderately enhanced with glucocorticoid treatment. Inclusion of adrenaline in the culture medium resulted in elevated incorporation of copper into newly synthesized caeruloplasmin as well as an increase in 64Cu-labelled caeruloplasmin in the culture medium. However, adrenaline did not seem to increase the secretion of 3H-labelled protein, despite the elevation in secreted 64Cu-caeruloplasmin. This may be due to a large increase in the intracellular pool of 64Cu caused by enhanced accumulation of this metal when adrenaline is included in the incubation medium. Enhanced copper accumulation was also seen when cells were treated with glucagon. Adrenaline-stimulated accumulation of 64Cu could be inhibited by including phenoxybenzamine, an alpha-adrenergic blocker, in the culture medium. Elevation of extracellular copper caused enhancement in the detection of labelled caeruloplasmin in the medium of cultured cells, probably owing to the ability of this metal to stabilize the protein.  相似文献   

4.
The inhibition of caeruloplasmin by azide   总被引:6,自引:6,他引:0  
1. The inhibition of the oxidase activity of caeruloplasmin by azide was investigated at 25 degrees and 7.5 degrees . 2. The inhibition is reversible on dilution or Sephadex treatment, indicating a caeruloplasmin-azide complex. 3. The enzyme is protected against azide inhibition by chloride, acetate or EDTA, the last-named acting not by chelation but by a non-specific effect similar to that of acetate. 4. Lineweaver-Burk plots with different concentrations of azide are parallel. This may occur either when the enzyme-substrate complex or when a subsequent intermediate structure of the enzyme forms the inhibited complex. 5. At 7.5 degrees inhibition may be shown not to occur until after the initial reaction of enzyme with substrate. 6. At 7.5 degrees , the inhibition is of the mutual-depletion type, inhibitory concentrations of azide being comparable with the concentration of caeruloplasmin. It is shown that the binding of a single azide group completely inhibits a caeruloplasmin molecule. 7. An arrangement of the four valence-changing copper atoms of caeruloplasmin is proposed in which they are so close together in the cuprous form that reoxidation may occur by the simultaneous transfer of four electrons from the copper atoms to a single oxygen molecule.  相似文献   

5.
1. The interpretation of the effects of mixtures of inhibitors on enzymes is considered. 2. The effects of inhibitor mixtures on caeruloplasmin were determined. 3. Fluoride, chloride and cyanate inhibit at one type of site (alpha), whereas bromide and iodide inhibit at another type (beta) present in the same enzyme intermediate. 4. Effects of inhibitor mixtures containing azide or cyanide are consistent with previous indications (Speyer & Curzon, 1968) that these ligands form inhibited complexes with different enzyme intermediates. 5. Isobols of halides or of cyanate with azide indicate that azide inhibits caeruloplasmin by bridging two alpha sites, these being reduced copper atoms. 6. Iodide and cyanate give hyperbolic plots of 1/v against [I]. 7. It is suggested that in the cyanate-inhibited complex the inhibitor binds to a reduced copper atom (alpha site) but that binding of cyanate at another copper atom is sterically prevented. It is suggested that the less bulky alpha-site inhibitors, fluoride and chloride, cause complete inhibition by binding to both of these copper atoms, which can also be bridged by a single azide group. 8. Each halide shows a pattern of effects on caeruloplasmin that is qualitatively distinct from that of other halides.  相似文献   

6.
The inhibition of caeruloplasmin by cyanide   总被引:3,自引:2,他引:1       下载免费PDF全文
1. The reversible inhibition of the oxidase activity of caeruloplasmin by cyanide was investigated. 2. The kinetics are unusual, being competitive but with the inhibited complex formed only during cycling. 3. Inhibitory concentrations of cyanide are comparable with that of caeruloplasmin. 4. One azide group completely inhibits a caeruloplasmin molecule but two cyanide groups are required. 5. The results suggest that azide binds to a half-reduced or fully reduced conformational isomer of the enzyme whereas cyanide binds to completely reoxidized isomers, and that inhibited complexes contain ligand bridges between copper atoms.  相似文献   

7.
The question of the stoichiometry of copper bound to dopamine beta-hydroxylase and the number of copper atoms required for maximal activity was addressed in this study. Incubation of tetrameric enzyme from bovine adrenal medulla with 64Cu2+ followed by rapid gel filtration yielded an enzyme containing 8.3-8.9 mol of Cu/mol of tetramer. An identical stoichiometry was obtained by analysis of bound copper by atomic absorption methods. NMR and EPR were used to monitor titrations of the enzyme with Cu2+ and showed that the longitudinal relaxation rate of solvent water protons and the amplitude of the signal at g approximately 2 increased linearly up to a copper to protein ratio of approximately 8. Additional titrations also indicate that an enzyme-Cu2+-tyramine-CN- inhibitory complex was formed when 8 mol of Cu2+ are bound per mol of enzyme. The rate of inactivation of dopamine beta-hydroxylase by the mechanism-based inhibitor 2-Br-3-(p-hydroxyphenyl)-1-propene was measured and used as a method to follow enzymatic catalysis. An increase in rate was observed with increasing Cu2+ up to a protein to Cu2+ ratio of 8 Cu/tetramer. The rate becomes constant after this ratio is achieved. These data indicate that dopamine beta-hydroxylase specifically binds 8 mol of Cu/tetramer and that this stoichiometry is required for maximal activity.  相似文献   

8.
Summary The release of intact CU(I)8-thionein from copper-resistant copper-loaded yeast cells, strain X 2180-1Aa, has been shown. This copper(I)-thiolate-rich protein was characterized and compared with the chemical and physicochemical properties of intracellular yeast Cu-thionein. The same molecular mass and stoichiometry of 8 mol copper atoms/mol protein was found. No detectable difference between the Cu-thioneins was seen in luminescence emission, electronic absorption in the ultraviolet region, chiroptical data or amino acid composition. The importance of stable Cu(I)-thiolates in Cu-thionein as a safe vehicle for transporting copper in a non-reactive manner is confirmed.  相似文献   

9.
The room-temperature e.p.r. spectrum of the Japanese-lacquer-tree (Rhus vernicifera) laccase shows A parallel (the hyperfine splitting constant) and g parallel values of both the Type 1 and Type 2 Cu appreciably different from those measured at liquid-N2 temperature. The geometry of the sites, as inferred from the room-temperature e.p.r. parameters, is more consistent with their redox properties. A rough correlation is found between A parallel and g parallel values and redox potential of the blue copper in several enzymes.  相似文献   

10.
Patients previously not treated with a lipid-lowering agent (n = 20; mean age 49.15 +/- 3.28 years) were treated with either 10 mg/day of Simvastatin (n = 11), or Atorvastatin (n = 9) for 4 months. Fourteen additional patients were recruited from the same clinic at the same hospital as a control group. The medication of these latter patients was unaltered for 4 months and the same parameters were measured as for the statin groups. Serum concentrations of zinc, copper, caeruloplasmin, selenium, glutathione peroxidase (GPx) and C-reactive protein (CRP) were measured together with their lipid profiles pre- and post-treatment. In addition to reducing serum total and low-density lipoprotein (LDL) cholesterol (p < 0.0001), statin treatment was associated with a significant reduction in mean serum zinc (9%, p = 0.03), copper (9%, p < 0.01), caeruloplasmin (24%, p < 0.05), and median CRP (45%, p < 0.03). Similar changes were not observed in the control patients. No significant effects were observed for serum selenium, copper/caeruloplasmin ratio, or GPx (p > 0.05) in either statin or control groups. These changes may be related to the known anti-inflammatory properties of the statin class of drugs.  相似文献   

11.
Ceruloplasmin from the turtle Caretta caretta was isolated to purity by using the single-step procedure recently developed by us to purify sheep and chicken ceruloplasmin. It has a Mr of ca. 145,000 and a total copper content of 5.1 +/- 0.2 atoms of copper per molecule, 50% of which are detectable by EPR. The spectroscopic features include an absorption maximum at 603 nm in the electronic spectrum and the total absence of any resonance attributable to Type 2 copper in the EPR spectrum. Turtle ceruloplasmin was found to be unusually resistant to aging and proteolysis, when compared to ceruloplasmins isolated from other species. p-Phenyl-endiamine oxidase activity measurements revealed an unusually low catalytic efficiency, while the kinetic parameters of Fe(II) oxidation were consistent with those reported for other species of ceruloplasmin.  相似文献   

12.
The interaction of one-electron reduced metronidazole (ArNO2.-) with native and Type-2-copper-depleted ascorbate oxidase were studied in buffered aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. With ArNO2.-, reduction of Type 1 copper of the native enzyme and of the Type-2-copper-depleted ascorbate oxidase occurs via a bimolecular step and at the same rate. Whereas the native protein accepts, in the absence of O2, 6-7 reducing equivalents, Type-2-copper-depleted ascorbate oxidase accepts only 3 reducing equivalents with stoichiometric reduction of Type 1 copper. On reaction of O2.- with ascorbate oxidase under conditions of [O2.-] much greater than [ascorbate oxidase], removal of Type 2 copper results in reduction of all the Type 1 copper atoms, in contrast with reduction of the equivalent of only one Type 1 copper atom in the holoprotein. From observations at 610 nm, the rate of reduction of ascorbate oxidase by O2.- is not dependent on the presence of Type 2 copper. For the holoprotein, no significant optical-absorption changes were observed at 330 nm. It is proposed that electrons enter the protein via Type 1 copper in a rate-determining step followed by a fast intramolecular transfer of electrons within the protein. For the Type-2-copper-depleted protein, intramolecular transfer within the protein, however, is slow or does not occur. In the presence of O2, it is also suggested that re-oxidation of the partially reduced holoprotein occurs at steady state, as inferred from the observations at 330 nm and 610 nm. The role of Type 2 copper in ascorbate oxidase is discussed in terms of its involvement in redistribution of electrons within the protein or structural considerations.  相似文献   

13.
The interactions of one-electron reduced metronidazole (ArNO2.-) and O2.- with native and Type-2-copper-depleted Vietnamese- and Japanese-lacquer-tree laccases were studied in aqueous solution at pH 6.0 and 7.4 by using the technique of pulse radiolysis. On reaction with ArNO2.-, in the absence of O2, the holo- and the Type-2-copper-depleted proteins accept, with reduction of Type 1 copper, 2 and 1 reducing equivalents respectively. On reaction with O2.- of both holo- and Type-2-copper-depleted Vietnamese-lacquer-tree laccase, almost complete reduction of Type 1 copper was observed and, after completion of the reaction, some (less than 20%) reoxidation of Type 1 copper occurs. Reduction of Type 1 copper of the laccases by these one-electron donors occurs via a bimolecular step; however, the rate of reduction of Vietnamese-lacquer-tree laccase is over 10 times that of Japanese-lacquer-tree laccase. It is inferred that electrons enter the protein via Type 1 copper with, in the case of the holoprotein, subsequent rapid intramolecular transfer of 1 reducing equivalent within the protein. Furthermore it is suggested that intra-molecular electron transfer to Type 3 copper atoms is slow and, in the case of Type-2-copper-depleted protein, may not occur. This slow process may partially account for the variation of the catalytic activities of 'blue' oxidases.  相似文献   

14.
The reactivity with dioxygen of a mammalian (sheep) ceruloplasmin, anaerobically reduced with ascorbate, was found to depend on the state of the Type 2 and Type 3 copper centers, as monitored by EPR and optical spectroscopy. A complete reoxidation by air after anaerobic reduction with ascorbate was observed with samples (A) purified by the single-step procedure described for chicken ceruloplasmin (Calabrese, L., Carbonaro, M., and Musci, G. (1988) J. Biol. Chem. 263, 6480-6483), while samples prepared by traditional multistep procedure (B) or subjected to freeze-thawing (C) displayed partial and very slow reoxidation, reflecting the functional nonequivalence of blue coppers which is considered a typical property of mammalian ceruloplasmin. The rate of reduction of the 330 nm chromophore was found to increase as a function of the extent and rate of reoxidation of different samples, while the 610 nm band displayed an opposite trend. Samples B and C showed a Type 2 copper signal in the EPR spectrum, while sample A showed practically no Type 2 copper in the oxidized protein, and a transient Type 2-like signal during reduction. The presence of a trinuclear Type 2-Type 3 cluster can therefore be proposed for all ceruloplasmins, and the integrity of the copper-copper coupling is essential for efficient oxidase behavior.  相似文献   

15.
Isolation of mannose-binding proteins from human and rat liver   总被引:7,自引:1,他引:7       下载免费PDF全文
The interaction of e-aq., CO2-. and one-electron reduced nitroaromatics (RNO2-.) with ascorbate oxidase (AAO) was studied in aqueous solution at pH 6.0 and 7.5 by using the technique of pulse radiolysis. From observations at 330, 410 and 610 nm, interaction of e-aq. and CO2-. with AAO results in non-specific reduction of the protein followed by reduction of Type 1 Cu in a rate-determining intramolecular step. Only a few per cent of the reducing equivalents ultimately results in reduction of Type 1 Cu. With large excesses of reducing equivalents (e-aq. and CO2-.) with respect to the copper concentration, the amount of Type 1 copper reduced never exceeds 50% of the total amount of Type 1 copper after a single radiation pulse. With less-powerful reducing agents, e.g. RNO2-. reduction of Type 1 Cu occurs via a bimolecular step, and there is no evidence for formation of radicals on protein residues. From observations at 330 nm it is evident that Type 2 and/or Type 3 Cu may also be reduced along with Type 1 Cu. Almost stoichiometric reduction of AAO by RNO2-. was observed, e.g. the protein accepts 6-7 reducing equivalents. It is inferred that the various types of redox couples Cu2+/Cu+ are in equilibrium and that intramolecular electron transfer between the different types of Cu is not rate-determining when using RNO2-. as reducing agent.  相似文献   

16.
Titration of cyanide-incubated cytochrome c oxidase (ox heart cytochrome aa3) with ferrocytochrome c or with NNN'N'-tetramethyl-p-phenylenediamine initially introduces two reducing equivalents per mol of cytochrome aa3. The first equivalent reduces the cytochrome a haem iron; the second reducing equivalent is not associated with reduction of the 830 nm chromophores (e.p.r.-detectable copper) but is probably required for reduction of the e.p.r.-undetectable copper. Excess reductant introduces a third reducing equivalent into the cyanide complex of cytochrome aa3. During steady-state respiration in the presence of cytochrome c and ascorbate, the 830 nm chromophore is almost completely oxidized. It is reduced more slowly than cytochrome a on anaerobiosis. In the presence of formate or azide, some reduction at 830 nm can be seen in the steady state; in an oxygen-pulsed system, a decrease in steady-state reduction of cytochromes c and a is associated with ab increased reduction of the 830 nm species. In the formate-inhibited system the reduction of a3 on anaerobiosis shows a lag phase, the duration of which corresponds to the time taken for the 830 nm species to be reduced. It is concluded that the e.p.r.-undetectable copper (CuD) is reduced early in the reaction sequence, whereas the detectable copper (CUD) is reduced late. The latter species is probably that responsible for reduction of the cytochrome a3 haem. The magnetic association between undetectable copper and the a3 haem may not imply capability for electron transfer, which occurs more readily between cytochrome a3 and the 830 nm species.  相似文献   

17.
1. Laccases I, II and III were (EC 1.14.18.1) prepared from the mycelium of the ascomycete Podospora anserina. The tetrameric laccase I(mol. wt 340 000, 16 copper atoms) and the monomeric laccases II and II (mol. wt 80 000, 4 copper atoms) have been studied by optical absorption-, circular dichroism-(CD)and electron paramagnetic resonance spectroscopy (EPR). 2. The visible and near ultraviolet difference absorption spectrum, which is apparently identical for all three laccases, shows two maxima at 330 and 610 nm and a shoulder at about 725 nm. The molar extinction coefficients of these bands are 4 times larger for the tetrameric laccase I compared to the monomeric laccases II and III which show values similar to other blue copper-containing oxidases. 3. CD spectra between 300 and 730 nm of the tree laccases are similar and contain at least 5-bands in the oxidized enzyme. If the enzyme is reduced, only a band at 307 nm remains. The molar ellipticity values of these bands are 4 times larger for laccase I than the corresponding bands of laccases II and III. It is inferred that the reducible bands are associated with the Type 1 Cu-2+. 4. In all three laccases the EPR-detectable copper accounts for only about 50% of the total copper content. The 9-GHz and 35-GHz spectra, which are identical for all three laccases, consist of two components of equal intensity. One component shows a rather small copper hyperfine coupling and a small deviation from axial symmetry. It is suggested that this copper is associated with the blue chromophore in analogy to Type 1 Cu-2+ in other blue copper proteins. The other component has a broader hyperfine coupling similar to Type 2 Cu-2+ as found in other copper proteins. The assumption that the experimental spectra result from a superposition of the spectra of equal amounts of Type 1 and Type 2 Cu-2+ has been verified by computer simulation. 5. It is suggested that the copper ions which are not detected by EPR are connected to the absorption band at 330 nm and that these ions are also essential for the function of these laccases.  相似文献   

18.
《Inorganica chimica acta》1988,151(4):265-268
Titration of apo-caeruloplasmin employing substoichiometric concentrations of [Cu(I)-(thiourea)3]Cl was performed to elucidate possible sequential incorporation of copper into the different specific binding sites. The successful reconstitution was monitored by A610 absorption, EPR spectroscopy and oxidase activity. Maximum activity and final absorption at 610 nm were reached after 20 min. When both A610, indicative for type 1 copper, and oxidase activity were expressed per g-atom of copper, a sequential insertion was found. Owing to the specific data at the beginning, some type 3 copper appeared to be preferentially incorporated. After 3–4 g-atoms (including most of type 1 and type 2 copper), both absorption and oxidase activity surpassed transient maxima. Then type 3 and 4 copper were further bound to reach the known stoichiometry of six copper atoms per mole of protein.  相似文献   

19.
1. Detailed studies on the mechanism of the enzymic reaction of bovine superoxide dismutase were carried out by using pulse radiolysis and electron paramagnetic resonance (e.p.r.). 2. The second-order rate constant for reaction between superoxide dismutase and the superoxide ion was redetermined as (2.37+/-0.18)x10(9)m(-1).s(-1) at 25 degrees C. This reaction governs the turnover, and any first-order steps must have rate constants higher than about 10(6)s(-1). Turnover has a low activation energy and is slowed substantially when the viscosity is increased with glycerol, confirming that the reaction rate is near the limit for diffusion control. In water a reversible conformation change to a less active form appears to take place above about 40 degrees C. 3. Pre-steady-state rates of reduction and reoxidation of copper in the enzyme are consistent with these processes being rate-limiting in enzyme turnover. 4. Examination, with the help of computer simulation, of the e.p.r. spectra at 9 and 35GHz of native superoxide dismutase indicated that, apart from 10-20% of impurities, only one species of Cu(2+) is distinguishable. Further, the specific activity of our enzyme preparations, measured by pulse radiolysis, is at least as high as that obtained by other workers. 5. Nevertheless, measurement of the proportion of copper present as Cu(2+) (determined both optically and by e.p.r. spectroscopy) in the steady states approached from both the oxidized and the reduced forms of the enzyme, indicates (after allowing for the impurities) that only half of the copper atoms participate in turnover. E.p.r. spectroscopy provided no evidence for differences between functioning and non-functioning Cu(2+) atoms. 6. It is suggested that the results may be best interpreted in terms of an allosteric type of mechanism, with two initially indistinguishable copper atoms in the enzyme. Reaction of one of these with a superoxide ion then renders the other, at least transiently, unreactive.  相似文献   

20.
1. The oxidation of NN-dimethyl-p-phenylenediamine (DPD) by inorganic oxidants and by caeruloplasmin was studied. Some experiments were also made with NNN'N'-tetramethyl-p-phenylenediamine (TPD). 2. E(mM) (550) of the first free radical oxidation product of DPD (DPD(+)) was 9.8 and E(mM) (563) of the corresponding product of TPD (TPD(+)) was 12.5. 3. The non-enzymic decomposition of DPD(+) was studied with respect to temperature, pH, concentration and DPD/DPD(+) ratio, thus defining conditions for enzyme experiments under which DPD(+) extinction at 550mmu was proportional to enzyme activity. 4. Rates of oxidation of DPD to DPD(+) by caeruloplasmin were constant over a range of DPD concentrations. At low DPD concentrations a lag period occurred, which was eliminated by addition of DPD(+). 5. A lag period was not observed with TPD, but at low TPD concentrations the rate of TPD(+) formation was greater when TPD(+) was added. This suggests that TPD(+) may compete weakly as a substrate with TPD and may be oxidized further by the enzyme before a non-enzymic reaction with TPD to form more TPD(+). 6. With DPD sulphate or acetate or TPD sulphate as substrate, Lineweaver-Burk plots were curved. With DPD hydrochloride the chloride ion caused inhibition at higher concentrations, opposing the curvature. 7. Curved Lineweaver-Burk plots were interpreted in terms of two types of substrate binding site with different K(m) values but similar V(max.) values. 8. The apparent thermodynamic changes associated with enzyme-substrate-complex formation at the sites with higher K(m) suggest that considerable conformational change may occur on binding at these sites. 9. With substrate concentrations at which only the low-K(m) sites are involved 2mol. of DPD(+)/mol. of caeruloplasmin are formed before a steady state is established. At higher substrate concentrations up to 3.2mol. of DPD(+)/mol. of caeruloplasmin are formed at this initial stage. 10. Results are discussed in relation to caeruloplasmin structures in which (a) two valence-changing and two permanently cuprous copper atoms are more accessible than the remaining four copper atoms or (b) binding of substrate at one site hinders access of substrate to another site.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号