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1.
Enriched cytochrome b558 preparations were obtained from human mature monocytes (MN) and retinoic acid plus interferon gamma induced human myeloid leukemia cell lines HL-60 and U937, using an adaptation of the procedure described by A.W. Segal (Nature (1987) 326, 88-91) for purification of cytochrome b558 from human polymorphonuclear leukocytes (PMN). Spectral characteristics of cytochrome b558 were determined and found to be independent of cell type and specific heme b content of the preparation. To increase the sensitivity of the spectral assay, analysis in the gamma band were used and delta epsilon 427-413 was determined to be equal to 158 mM-1 cm-1. An alpha beta type heterodimeric cytochrome b558 was found for PMN and MN by the concordant elution of heme b spectral activity from heparin agarose and the detection of two polypeptide chains by SDS-PAGE. The expression of the lighter polypeptide alpha chain in the various human monocyte-like cell lines was assessed and its identity, as a component of cytochrome b, was confirmed by immunodetection using a rabbit polyclonal antibody reacting with the alpha subunit of PMN cytochrome b558. Immunoblotting studies detected the alpha subunit in monocyto-macrophagic differentiated HL-60 and U 937 cells and mature MN at 22 kDa, but not in uninduced cells which did not express the respiratory burst. Whatever the specific content or the cell origin of the cytochrome b558-enriched preparations, the heme b binding site was shown to be associated with the alpha subunit defined by a constant molecular mass of 22 kDa, as evidenced by the finding of a constant ratio between the silver stained band intensity and the corresponding heme b amount. The heavy polypeptide beta chain from MN cytochrome b was found to have a significantly higher molecular weight than the beta subunit from PMN at 94 +/- 5 kDa instead of 90 +/- 4 kDa. In contrast, in cytochrome b preparations from induced monocyto-macrophagic cells, isolated with a low heme specific content, the variability in the detection of the staining intensity of the beta band either in SDS-PAGE or immunodetection reactivities precludes accurate definition of its molecular mass and estimation of the stoichiometry between the alpha and beta subunits in the differentiated cells. However, wheat-germ agglutinin binding studies indicated the presence of N-glycosylated protein in the range of 85-110 kDa. 相似文献
2.
M Prats 《European journal of biochemistry》1977,75(2):619-625
The L-(+)-Lactate:cytochrome c oxidoreductase or cytochrome b2 from the yeasts Saccharomyces cerevisiae and Hansenula anomala were partially hydrolysed in various concentrations of trypsin. Conditions were found which allowed the isolation from the Hansenula enzyme of a 140 000 +/- 10 000-dalton flavoprotein. The prosthetic flavin groups were still reducible by substrate (spectroscopic evidence) but the flavoprotein was unable to form a complex with cytochrome c, the physiological acceptor in the enzymatic reaction. No such flavoprotein units could be found during proteolysis of the Saccharomyces enzyme. The heme prosthetic group of the Hansenula enzyme remained bound to a 15 500 +/- 1000-dalton protein unit which was larger than, but very similar to, the well known 'cytochrome b2 core' of the Saccharomyces enzyme. Moreover, the degradation of different enzyme samples by contaminated proteases allowed the isolation of a particular form of Hansenula enzyme: each tetramer had, on the mean, four bound flavins and only two heme groups. These molecules completely retained their ability to form a complex with cytochrome c. 相似文献
3.
Iron and probably also copper are absorbed by the intestine in their reduced form. A b-type cytochrome, Dcytb, has recently been cloned from mouse and has been proposed to be the corresponding reductase. However, the nature of the cytochrome and the reduction reaction remain unknown. Here we describe the isolation and functional characterization of a novel b-type cytochrome from rabbit enterocytes. The 33 kDa heme protein was solubilized from brush border membranes with Triton X-100 and purified by successive ion exchange chromatography and hydrophobic interaction chromatography. Spectroscopic analysis of the heme revealed a b(558) cytochrome. The purified hemoprotein exhibited ascorbate-stimulated reduction of iron(III) and copper(II). The rate constants, k(1), for these reactions were 1.38 +/- 0.12 and 0.64 +/- 0.16 min(-1), respectively. Cytochrome b(558) may be the rabbit Dcytb homologue. A novel mechanism of how cytochrome b(558) could shuttle electrons from cytoplasmic ascorbate to luminal dehydroascorbate is proposed. 相似文献
4.
Cláudio M. Gomes Arnulf Kletzin Miguel Teixeira 《Journal of biological inorganic chemistry》2002,7(4-5):483-489
A new type of cytochrome b was isolated from the cytoplasmatic fraction of the archaeon Acidianus ambivalens, which is the first soluble cytochrome found in this member of the thermoacidophilic order of the Sulfolobales. The protein is a monomeric and monohemic cytochrome b with a molecular mass of 22 kDa. Visible spectroscopy of the as-purified protein shows a Soret peak at 405 nm and a broad band at 625 nm, indicating the presence of a high-spin ferric heme. Upon reduction, the Soret band shifts to 422 nm and a broad band at 560 nm develops, again characteristic of high-spin ferrous heme. The reduced form can bind carbon monoxide, with visible absorption bands arising at 411 and 566 nm. EPR spectroscopy of the oxidized protein shows a spectrum typical of a high-spin heme, with major g values at 6.56 and 5.85. The reduction potential of the heme cofactor was determined to be -16+/-10 mV, at pH 6.5. Analysis of the protein amino acid sequence shows that it consists of a novel arrangement of domains. The first domain, at the N-terminus, has a remarkable similarity towards beta class carbonic anhydrases, whereas the second region comprises a putative cytochrome domain. The latter presumably consists of a novel fold, as it bears no sequence similarities towards other known cytochromes, or towards known domains. Strikingly, the first module contains the C-X (n)-H-X(2)-C motif that accounts for the binding of the catalytic zinc in carbonic anhydrases, but lacks several other critical residues required for substrate binding and proper active site geometry. In agreement with this finding, the isolated cytochrome contains one bound zinc atom, but has no carbonic anhydrase activity. Inspection of the sequences available from the genomic sequencing project of the close relative archaeon Sulfolobus solfataricus shows the presence of an identical protein, suggesting its dissemination among the Sulfolobales. The role of zinc as a key element for the intrinsic thermal stability of these proteins is discussed. 相似文献
5.
Inmaculada Yruela Francisca Miota Elena Torrado Michael Seibert Rafael Picorel 《European journal of biochemistry》2003,270(10):2268-2273
The cytochrome b559 content was examined in five types of isolated photosystem II D1-D2-cytochrome b559 reaction center preparations containing either five or six chlorophylls per reaction center. The reaction center complexes were obtained following isolation procedures that differed in chromatographic column material, washing buffer composition and detergent concentration. Two different types of cytochrome b559 assays were performed. The absolute heme content in each preparation was obtained using the oxidized-minus-reduced difference extinction coefficient of cytochrome b559 at 559 nm. The relative amount of D1 and cytochrome b559alpha-subunit polypeptide was also calculated for each preparation from immunoblots obtained using antibodies raised against the two polypeptides. The results indicate that the cytochrome b559 heme content in photosystem II reaction center complexes can vary with the isolation procedure, but the variation of the cytochrome b559alpha-subunit/D1 polypeptide ratio was even greater. This variation was not found in the PSII-enriched membrane fragments used as the RC-isolation starting material, as different batches of membranes obtained from spinach harvested at different seasons of the year or those from sugar beets grown in a chamber under controlled environmental conditions lack variation in their alpha-subunit/D1 polypeptide ratio. A precise determination of the ratio using an RC1-control sample calibration curve gave a ratio of 1.25 cytochrome b559alpha-subunit per 1.0 D1 polypeptide in photosystem II membranes. We conclude that the variations found in the reaction center preparations were due to the different procedures used to isolate and purify the different reaction center complexes. 相似文献
6.
The backbone dynamics of ferricytochrome b(562), a four-helix bundle protein from Escherichia coli, have been studied by NMR spectroscopy. The consequences of the introduction of a c-type thioether linkage between the heme and protein and the reduction to the ferrous cytochrome have also been analyzed. (15)N relaxation rates R(1) and R(2) and (1)H-(15)N NOEs were measured at proton Larmor frequencies of 500 and 600 MHz for the oxidized and reduced protein as well as for the oxidized R98C variant. In the latter protein, an "artificial" thioether covalent bond has been introduced between the heme group and the protein frame [Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., de Lumley Woodyear, T., Johnson, C. M., and Barker, P. D. (2000) Biochemistry 39, 1499-1514]. The (15)N relaxation data were analyzed with the ModelFree protocol, and the mobility parameters on the picosecond to nanosecond time scale were compared for the three species. The three forms are rather rigid as a whole, with average generalized order parameters values of 0.87 +/- 0.08 (oxidized cytochrome b(562)), 0.84 +/- 0.07 (reduced cytochrome b(562)), and 0.85 +/- 0.07 (oxidized R98C cytochrome b(562)), indicating similar mobility for each system. Lower order parameters (S(2)) are found for residues belonging to loops 1 and 2. Higher mobility, as indicated by lower order parameters, is found for heme binding helices alpha 1 and alpha 4 in the R98C variant with respect to the wild-type protein. The analysis requires a relatively long rotational correlation time (tau(m) = 9.6 ns) whose value is accounted for on the basis of the anisotropy of the molecular shape and the high phosphate concentration needed to ensure the occurrence of monomer species. A parallel study of motions in the millisecond to microsecond time scale has also been performed on oxidized wild-type and R98C cytochrome b(562). In a CPMG experiment, decay rates were analyzed in the presence of spin-echo pulse trains of variable spacing. The dynamic behavior on this time scale is similar to that observed on the sub-nanosecond time scale, showing an increased mobility in the residues connected to the heme ligands in the R98C variant. It appears that the increased protein stability of the variant, established previously, is not correlated with an increase in rigidity. 相似文献
7.
Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase 总被引:1,自引:0,他引:1
Barber MJ Desai SK Marohnic CC Hernandez HH Pollock VV 《Archives of biochemistry and biophysics》2002,402(1):38-50
Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme. 相似文献
8.
Steensma E Gordon E Oster LM Ferguson SJ Hajdu J 《The Journal of biological chemistry》2001,276(8):5846-5855
The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the c heme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met106 has left the heme iron. Melting studies show that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1.2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of the d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed. 相似文献
9.
The ba3 cytochrome c oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS, and FTIR spectroscopic approach. Oxidative electrochemical redox titrations yielded midpoint potentials of Em1= -0.02 +/- 0.01 V and Em2 = 0.16 +/- 0.04 V for heme b and Em1 = 0.13 +/- 0.04 V and Em2 = 0.22 +/- 0.03 V for heme a(3) (vs Ag/AgCl/3 M KCl). Fully reversible electrochemically induced UV/VIS and FTIR difference spectra were obtained for the full potential step from -0. 5 to 0.5 V as well as for the critical potential steps from -0.5 to 0.1 V (heme b is fully oxidized and heme a3 remains essentially reduced) and from 0.1 to 0.5 V (heme b remains oxidized and heme a3 becomes oxidized). The difference spectra thus allow to us distinguish modes coupled to heme b and heme a3. Analogous difference spectra were obtained for the enzyme in D2O buffer for additional assignments. The FTIR difference spectra reveal the reorganization of the polypeptide backbone, perturbations of single amino acids and of hemes b and a3 upon electron transfer to/from the four redox-active centers heme b and a3, as well as CuB and CuA. Proton transfer coupled to redox transitions can be expected to manifest in the spectra. Tentative assignments of heme vibrational modes, of individual amino acids, and of secondary structure elements are presented. Aspects of the uncommon electrochemical and spectroscopic properties of the ba3 oxidase from T. thermophilus are discussed. 相似文献
10.
Human phagocyte cytochrome b is the terminal component of the microbicidal superoxide generating system. Although the primary structure of this protein has been determined, little is known about the placement of the heme prosthetic groups in this heterodimeric integral membrane protein. Analysis of the cytochrome using lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 0 degree C followed by tetramethylbenzidine heme staining demonstrated the presence of heme in both the 91- and 22-kDa subunits identified by Western blot analysis using peptide specific antisera. Exposure of cytochrome b (purified or in isolated neutrophil plasma membranes) to Staphylococcal protease V8 or trypsin did not affect absorbance spectra. However, such treatment resulted in degradation of both subunits to smaller fragments, including characteristic immunoreactive 20-kDa fragments of both the large and small subunits of the cytochrome that retained one or both of the hemes. The spectral stability to proteolysis and size of the proteolytic heme-containing fragments generated explains previous reports which suggested that the heme resided in the small subunit. Our current results indicate that human neutrophil cytochrome b is a bi-heme or possibly tri-heme molecule with at least one heme residing in the large subunit and one shared between both subunits and that the heme-containing regions of the cytochrome probably lie within the membrane lipid bilayer. Such a multi-heme structure would be consistent with an electron transfer function for this cytochrome by providing an efficient mechanism for transferring electrons across the plasma membrane to the extracellular surface where oxygen could be reduced to create superoxide. 相似文献
11.
On the question of interheme electron transfer in the chloroplast cytochrome b6 in situ 总被引:1,自引:0,他引:1
The redox properties, the site of action of the inhibitor NQNO, and the question of interheme transfer in the chloroplast cytochrome b6 have been examined with regard to the role of the b6-f complex in quinol oxidation and H+ translocation. (i) The two hemes of the cytochrome ba and bp, have similar (delta Em less than or equal to 50 mV) oxidation-reduction midpoint potentials that are pH-independent in the range pH 6.5-8.0 (Em7 = -40 mV) but are pH dependent below this range with an estimated pK = 6.7. (ii) Only half of cytochrome b6, the stromal-side heme, ba, was reducible by NADPH and ferredoxin. (iii) The 2-3-fold increase (to 0.60 +/- 0.09 heme/600 Chl) in the amplitude of flash-induced cytochrome reduction caused by NQNO was not affected when heme ba was initially reduced, implying that NQNO affects flash reduction at the site of heme bp. (iv) Multiple light flashes did not increase the amplitude of b6 reduction in the presence or absence of NQNO or show binary oscillations. Together with localization of a site of action of NQNO near heme bp, these data provide no evidence for efficient electron transfer from heme bp to heme ba as specified by the Q cycle model. (v) NQNO interaction with heme bp does not block its oxidation, since reoxidation of the flash-reduced cytochrome in its presence or absence was 4-5 times faster (t1/2 approximately 30 ms) when heme ba was reduced. The faster oxidation of the photoreduced cytochrome after NADPH-Fd reduction of heme ba indicates that the oxidation of ba and bp may be cooperative. 相似文献
12.
B D Sidell 《The Journal of experimental zoology》1977,199(2):233-250
The concentration of cytochrome c in the skeletal muscle of the green sunfish (Lepomis cyanellus) increases with decreasing temperature of acclimation: 1.51 +/- 0.09, 1.17 +/- 0.03, and 0.98 +/- 0.07 nanomoles per gram wet weight from muscle of animals acclimated to 5 degrees, 15 degrees, and 25 degrees C, respectively. The roles of synthesis and degradation of cytochrome c during thermal acclimation were investigated by measurement of loss of specific radioactivity from cytochrome c and from total mitochondrial heme protein, and by analysis of the rate of change in concentration of cytochrome c. The radioisotope used was 14C-delta-aminolevulinic acid, a non-reutilizable heme precursor. At 25 degrees C, the half-life of cytochrome c was 7.1 days based on radioactivity measurements and 5.6 days based on change in concentration. Statistical analysis showed no significant difference in half-lives obtained by the two methods. The half-life of total mitochondrial heme protein was determined to be 5.7 days on the basis of radioactivity data, under the same conditions. No significant difference was found between the rate of turnover of the heme protein pool from mitochondria and either measurement for cytochrome c at 25 degrees C. At an acclimation temperature of 5 degrees C, the half-life of cytochrome c from skeletal muscle was 13.7 days based upon changes in concentration. At low acclimation temperature, radioactive label was retained in acid-soluble form by fish for many days, precluding measurement of half-life by this technique. Transfer of fish from 25 degrees to 5 degrees C resulted in a rapid decrease of approximately 40% in rates in synthesis of skeletal muscle cytochrome c, and a concomitant decrease in the degradation rate constant for this molecule of approximately 60%. The disproportionality in temperature-sensitivities of these two processes leads to an approximately 50% net increase in the concentration of cytochrome c during acclimation. In transfer from 5 degrees to 25 degrees C, the converse, rapid readjustments in synthetic and degradative parameters occur, resulting in the observed decrease in cytochrome c content. 相似文献
13.
The de novo design and synthesis of ruthenium-labeled cytochrome b5 that is optimized for the measurement of intracomplex electron transfer to cytochrome c are described. A single cysteine was substituted for Thr-65 of rat liver cytochrome b5 by recombinant DNA techniques [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. The single sulfhydryl group on T65C cytochrome b5 was then labeled with [4-(bromomethyl)-4'-methylbipyridine] (bisbipyridine)ruthenium2+ to form Ru-65-cyt b5. The ruthenium group at Cys-65 is only 12 A from the heme group of cytochrome b5 but is not located at the binding site for cytochrome c. Laser excitation of the complex between Ru-65-cyt b5 and cytochrome c results in electron transfer from the excited state Ru(II*) to the heme group of Ru-65-cyt b5 with a rate constant greater than 10(6) s-1. Subsequent electron transfer from the heme group of Ru-65-cyt b5 to the heme group of cytochrome c is biphasic, with a fast-phase rate constant of (4 +/- 1) x 10(5) s-1 and a slow-phase rate constant of (3 +/- 1) x 10(4) s-1. This suggests that the complex can assume two different conformations with different electron-transfer properties. The reaction becomes monophasic and the rate constant decreases as the ionic strength is increased, indicating dissociation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
Sone N Nagata K Kojima H Tajima J Kodera Y Kanamaru T Noguchi S Sakamoto J 《Biochimica et biophysica acta》2001,1503(3):279-290
Electrophoresis of a Corynebacterium glutamicum membrane preparation in the presence of sodium dodecyl sulfate, followed by staining for peroxidase activity (heme staining), showed only one band at about 28 kDa. This 28 kDa protein was purified from C. glutamicum membranes by chromatography in the presence of decylglucoside using DEAE-Toyopearl and hydroxylapatite columns, as the sole c-type cytochrome in the bacterium. The cytochrome showed an alpha band at 551 nm, and its E(m, 7) was about 210 mV. A QcrCAB operon encoding the subunits of a putative quinol cytochrome c reductase was found 3'-downstream of ctaE encoding subunit III of cytochrome aa(3) in the C. glutamicum genome. The deduced amino acid sequence of qcrC, composed of 283 amino acid residues, contained two heme C-binding motifs and was in agreement with partial peptide sequences obtained from the 28 kDa protein after V8 protease digestion. We propose to name this protein cytochrome cc. The presence of cytochrome cc is a common feature of high G+C content Gram-positive bacteria, since we could confirm this protein by electrophoresis; homologous QcrCAB operons are also known in Mycobacterium and Streptomyces. QcrA and qcrB of C. glutamicum encode the Rieske Fe-S protein and cytochrome b, respectively, although these proteins were not co-purified with cytochrome cc. The phylogenetic tree of cytochromes b and b(6) show that C. glutamicum cytochrome b, along with those of other bacteria in the high G+C group, is rather different from the Bacillus counterparts, but highly similar to the Deinococci and Thermus cytochromes. This indicates that there is a fourth group of bacteria in addition to the three clades: proteobacterial cytochrome b, cyanobacterial b(6) and green sulfur-low G+C Gram-positive bacteria. 相似文献
15.
Spectroelectrochemical studies are presented for the carbon monoxide complex of isolated, purified cytochrome c oxidase (EC 1.9.3.1) in solutions saturated with carbon monoxide. The results indicate a stoichiometry of three equivalents per oxidase-carbon monoxide complex molecule. Formal reduction potentials (Eo) of the two copper and one heme component at pH 7.0 were obtained by means of quantitative absorbance-charge titrations in the absence and presence of cytochrome c, and by means of a Nernstian "Minnaert" plot in the presence of cytochrome c. Analysis of the absorbance-charge curves from these titrations gave an indirect determination of the high potential, "invisible" copper component. The copper potentials in the carbon monoxide complex were found to be relatively unchanged with respect to those of the native enzyme. The Eo values obtained were: high potential ("invisible") copper (340 +/- 20 mV (NHE)), low potential copper (190 +/- 20 mV), and low potential heme (250 +/- 10 mV). 相似文献
16.
Using surface-modified electrodes composed of omega-hydroxyalkanethiols, an experimentally based value for the inner-sphere reorganization energy of the bis(imidazole)iron porphyrin system has been obtained by examining the solvent dependence of the reorganization energy of bis(N-methylimidazole)meso-tetraphenyl iron porphyrin. The value obtained (0.41 +/- 0.06 eV) is remarkably similar to values we have recently reported for the reorganization energy of cytochrome b(5) (0.43 +/- 0.02 eV) and cytochrome c (0.58 +/- 0.06 eV). This strongly suggests that the protein matrix mimics the behavior of a low dielectric solvent and effectively shields the heme from the solvent. The effect of the orientation of the heme relative to the electrode was also explored by sytematically varying the steric bulk of the axial ligands. On the basis of a good linear correlation between the electronic coupling and the cosine of the angle between the heme plane and the surface of the electrode, it is suggested that a parallel orientation of the heme yields a maximum in the electronic coupling. Relevance to interheme protein electron transfer is discussed. 相似文献
17.
The spin state of the heme in superoxide (O(2)(.)(-))-producing cytochrome b(558) purified from pig neutrophils was examined by means of room-temperature magnetic circular dichroism (MCD) under physiological conditions. Cytochrome b(558) with varying amounts of low-spin and high-spin heme was prepared by either pH adjustment or heat treatment, and the O(2)(.)(-)-forming activity in a cell-free system was found to correlate with the low-spin heme content. The possibility that the O(2)(.)(-)-forming activity results from a transient high-spin ferric heme form that is induced during activation by anionic amphophils has also been investigated. EPR spectra of cytochrome b(558) activated by either arachidonic acid or myristic acid, showed that a transient high-spin ferric species accounting for approximately 50% of the heme appeared in the presence of arachidonic acid, but not in the presence of myristic acid. Hence the appearance of a transient high-spin ferric heme species on activation with an amphophil does not afford a common activation mechanism in the NADPH oxidase system. The EPR results for cytochrome b(558) activated with arachidonic acid showed that the transient high-spin ferric heme can bind cyanide. However, the high-spin ferric heme does not contribute to the O(2)(.)(-) production of cytochrome b(558) in cell-free assays in the presence of cyanide. 相似文献
18.
Tomo T Akimoto S Tsuchiya T Fukuya M Tanaka K Mimuro M 《Photosynthesis research》2008,98(1-3):293-302
We isolated highly-purified photochemically active photosystem (PS) II reaction center (RC) complexes from the cyanobacterium Synechocystis sp. PCC 6803 using a histidine-tag introduced to the 47 kDa chlorophyll protein, and characterized their spectroscopic properties. Purification was carried out in a one-step procedure after isolation of PS II core complex. The RC complexes consist of five polypeptides, the same as in spinach. The pigment contents per two molecules of pheophytin a were 5.8 +/- 0.3 chlorophyll (Chl) a and 1.8 +/- 0.1 beta-carotene; one cytochrome b(559) was found per 6.0 Chl a molecules. Overall absorption and fluorescence properties were very similar to those of spinach PS II RCs; our preparation retains the best properties so far isolated from cyanobacteria. However, a clear band-shift of pheophytin a and beta-carotene was observed. Reasons for these differences, and RC composition, are discussed on the basis of the three-dimensional structure of complexes. 相似文献
19.
Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state. 相似文献
20.
A 110 fold purification of cytochrome b558 from resting bovine neutrophils has been achieved. The plasma membrane bound cytochrome b was extracted with aminoxide WS35, a non ionic detergent. The purification procedure included liquid column chromatography on CM-C50 Sephadex, chromatofocusing on the anion exchanger PBE94, and gel filtration on P30 Biogel. The purified preparation was characterized by an heme to protein (nmol/mg) ratio of 7.7. The isoelectric point of cytochrome b was at pH 6.5. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate three bands corresponding to apparent Mr 64,000, 56,000 and 20,000 were revealed by staining with Coomassie Blue. 相似文献