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1.
Arabidopsis thaliana 《Plant Physiology and Biochemistry》2000,38(12):913
Recent advances in the Arabidopsis sequencing project has elucidated the presence of two genes Atb561-A and Atb561-B that show limited homology to the DNA sequence encoding for the mammalian chromaffin granule cytochrome b-561 (cyt b-561). Detailed analysis of the structural features and conserved residues reveals, however, that the structural homology between the presumptive Arabidopsis proteins and the animal proteins is very high. All proteins are hydrophobic and show highly conserved transmembrane helices. The presumably heme-binding histidine residues in the plant and animal sequences as well as the suggested binding site for the electron acceptor, monodehydroascorbate, are strictly conserved. In contrast, the suggested electron donor (ascorbate) binding site is not very well conserved between the plant and animal sequences questioning the function of this motif. Sequence analysis of the Atb561-B gene demonstrates a different splicing than that initially predicted in silico resulting in a protein with nine extra amino acids and a significantly higher homology to the other cyt b-561 sequences. The homology between the plant and animal sequences is further supported by the strong similarity between a number of biochemical properties of the chromaffin cyt b-561 and the cyt b-561 isolated from bean hook plasma membranes. Since the mammalian cyt b-561 is considered specific to neuroadrenergic tissues, the identification of a closely related homologue in an aneural organism demonstrates that these proteins constitute a new class of widely occurring membrane proteins. Both the plant and animal cyt b-561 are involved in transmembrane electron transport using ascorbate as an electron donor. The similarity between these proteins therefore suggests, for the first time, that this transport supports a number of different cell physiological processes. An evolutionary relationship between the plant and animal proteins is presented. 相似文献
2.
Lucia Cenacchi Manuela Busch Philipp G. Schleidt Florian G. Müller Tina V.M. Stumpp Werner Mäntele Paolo Trost C. Roy D. Lancaster 《生物化学与生物物理学报:生物膜》2012,1818(3):679-688
Cytochrome (cyt) b561 proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b561-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b561 paralogs from Arabidopsis thaliana (Acytb561-A, Acytb561-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb561-A resembles the best characterised member of the CYBASC family, the cytochrome b561 from adrenomedullary chromaffin vesicles, and that Acytb561-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (EM) values were found to be fully consistent with ascorbate oxidation activities and Fe3 +-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b561 from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem EM values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem EM values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe3 +-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem EM values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b561 paralogs exist as homodimers. 相似文献
3.
Summary During the past twenty years evidence has accumulated on the presence of a specific high-potential, ascorbate-reducibleb-type cytochrome in the plasma membrane (PM) of higher plants. This cytochrome is named cytochromeb
561 (cytb
561) according to the wavelength maximum of its -band in the reduced form. More recent evidence suggests that this protein is homologous to ab-type cytochrome present in chromaffin granules of animal cells. The plant and animal cytochromes share a number of strikingly similar features, including the high redox potential, the ascorbate reducibility, and most importantly the capacity to transport electrons across the membrane they are located in. The PM cytb
561 is found in all plant species and in a variety of tissues tested so far. It thus appears to be a ubiquitous electron transport component of the PM. The cytochromesb
561 probably constitute a novel class of transmembrane electron transport proteins present in a large variety of eukaryotic cells. Of particular interest is the recent discovery of a number of plant genes that show striking homologies to the genes coding for the mammalian cytochromesb
561. A number of highly relevant structural features, including hydrophobic domains, heme ligation sites, and possible ascorbate and monodehydroascorbate binding sites are almost perfectly conserved in all these proteins. At the same time the plant gene products show interesting differences related to their specific location at the PM, such as potentially N-linked glycosylation sites. It is also clear that at least in several plants cytb
561 is represented by a multigene family. The current paper presents the first overview focusing exclusively on the plant PM cytb
561, compares it to the animal cytb
561, and discusses the possible physiological function of these proteins in plants.Abbreviations Asc
ascorbate
- cyt
cytochrome
- DHA
dehydroascorbate
- E0
standard redox potential
- EST
expressed sequence tag
- His
histidine
- MDA
monodehydroascorbate
- Met
methionine
- PM
plasma membrane 相似文献
4.
Summary. Atomic models possessing the common structural features identified for the cytochrome b
561 (cyt b
561) protein family are presented. A detailed and extensive sequence analysis was performed in order to identify and characterize
protein sequences in this family of transmembrane electron transport proteins. According to transmembrane helix predictions,
all sequences contain 6 transmembrane helices of which 2–6 are located closely in the same regions of the 26 sequences in
the alignment. A mammalian (Homo sapiens) and a plant (Arabidopsis thaliana) sequence were selected to build 3-dimensional structures at atomic detail using molecular modeling tools. The main structural
constraints included the 2 pairs of heme-ligating His residues that are fully conserved in the family and the lipid-facing
sides of the helices, which were also very well conserved. The current paper proposes 3-dimensional structures which to our
knowledge are the first ones for any protein in the cyt b
561 family. The highly conserved His residues anchoring the two hemes on the cytoplasmic side and noncytoplasmic side of the
membrane are in all proteins located in the transmembrane helices 2, 4 and 3, 5, respectively. Several highly conserved amino
acids with aromatic side chain are identified between the two heme ligation sites. These residues may constitute a putative
transmembrane electron transport pathway. The present study demonstrates that the structural features in the cyt b
561 family are well conserved at both the sequence and the protein level. The central 4-helix core represents a transmembrane
electron transfer architecture that is highly conserved in eukaryotic species.
Received May 12, 2002; accepted September 20, 2002; published online May 21, 2003
RID="*"
ID="*" Correspondence and reprints: Institute of Biophysics, Biological Research Centre, P.O. Box 521, 6701 Szeged, Hungary.
E-mail: tpali@nucleus.szbk.u-szeged.hu 相似文献
5.
Summary Cytochromeb
561 (cytb
561) is a trans-membrane cytochrome probably ubiquitous in plant cells. In vitro, it is readily reduced by ascorbate or by juglonol, which in plasma membrane (PM) preparations from plant tissues is efficiently produced by a PM-associated NAD(P)Hquinone reductase activity. In bean hypocotyl PM, juglonol-reduced cytb
561 was not oxidized by hydrogen peroxide alone, but hydrogen peroxide led to complete oxidation of the cytochrome in the presence of a peroxidase found in apoplastic extracts of bean hypocotyls. This peroxidase active on cytb
561 was purified from the apoplastic extract and identified as an ascorbate peroxidase of the cytosolic type. The identification was based on several grounds, including the ascorbate peroxidase activity (albeit labile), the apparent molecular mass of the subunit of 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the dimeric native structure, the typical spectral properties of a heme-containing peroxidase, and an N-terminal sequence strongly conserved with cytosolic ascorbate peroxidases of plants. Cytb
561 used in the experiments was purified from bean hypocotyl PM and juglonol was enzymatically produced by recombinant NAD(P)H:quinone reductase. It is shown that NADPH, NAD(P)H:quinone reductase, juglone, cytb
561, the peroxidase interacting with cytb
561, and H2O2, in this order, constitute an artificial electron transfer chain in which cytb
561 is indirectly reduced by NADPH and indirectly oxidized by H2O2.Abbreviations APX
ascorbate peroxidase
-
b
561PX
cytochrome 6561 peroxidase
- CPX
coniferol peroxidase
- cyt
cytochrome
- GPX
guaia-col peroxidase
- IWF
intercellular washing fluid
- MDHA
monodehydroascorbate
- PM
plasma membrane 相似文献
6.
Cenacchi L Busch M Schleidt PG Müller FG Stumpp TV Mäntele W Trost P Lancaster CR 《Biochimica et biophysica acta》2012,1818(3):679-688
Cytochrome (cyt) b(561) proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b(561)-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b(561) paralogs from Arabidopsis thaliana (Acytb(561)-A, Acytb(561)-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb(561)-A resembles the best characterised member of the CYBASC family, the cytochrome b(561) from adrenomedullary chromaffin vesicles, and that Acytb(561)-B is atypical compared to other CYBASC proteins. Haem oxidation-reduction midpoint potential (E(M)) values were found to be fully consistent with ascorbate oxidation activities and Fe(3+)-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b(561) from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E(M) values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E(M) values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe(3+)-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E(M) values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b(561) paralogs exist as homodimers. 相似文献
7.
Adrenal cytochrome b561 (cyt b561), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b561 (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31-40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (bH) peak were seen with mutation of His92; the largest changes in the low-potential (bL) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g = 3.1 signal (bH) but not the g = 3.7 signal (bL). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the bH transition; mutations in His110 produced the largest decreases in ΔA561 for the bL transition. These results indicate that His92 can be considered part of the bH heme center, and His110 part of the bL heme center, in adrenal cyt b561. 相似文献
8.
Summary. The plant plasma membrane (PM) contains more than one b-type cytochrome. One of these proteins has a rather high redox potential (can be fully reduced by ascorbate) and is capable
of transporting electrons through the PM. Four genes encoding proteins with considerable homology to the sequences of cytochrome
b
561 proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b
561 located in the Arabidopsis PM, first PM vesicles were purified by aqueous polymer two-phase partitioning from the leaves
of 9-week-old A. thaliana. PM proteins were solubilized by nonionic detergent, and the fully ascorbate-reducible b-type cytochrome was partially purified by anion-exchange chromatography steps. Potentiometric redox titration of the fraction,
containing the fully ascorbate-reducible b-type cytochrome after the first anion-exchange chromatography step, revealed the presence of two hemes with redox potentials
of 135 mV and 180 mV, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing
the fully ascorbate-reducible b-type cytochrome after the second anion-exchange chromatography step revealed the presence of a single polypeptide band at
about 120 kDa. However, heat treatment (15 min, 90 °C) before electrophoresis was able to split the 120 kDa band into two
bands with molecular masses of about 23 and 28 kDa. These values are lower than the apparent molecular mass for the fully
ascorbate-reducible b-type cytochrome purified from Phaseolus vulgaris hypocotyls (about 52 kDa) but are in good agreement with those characteristic for the cytochrome b
561 proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis
genome (24–31 kDa).
Received May 4, 2002; accepted July 26, 2002; published online May 21, 2003
RID="*"
ID="*" Correspondence and reprints: Institute of Biophysics, BRC, Hungarian Academy of Sciences, POB 521, 6701 Szeged, Hungary. 相似文献
9.
Two membrane-bound, ascorbate-dependent b-type cytochromes were identified in etiolated bean (Phaseolus vulgaris L.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pH 8.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The -band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and behaved as a monomer of about 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes [P. Trost et al. (2000) Biochim Biophys Acta 1468:1–5]. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS–PAGE gels, was dimeric (60 kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochrome b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochrome b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochrome b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochrome b561 of animal chromaffin granules. The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis. 相似文献
10.
Summary Higher plant plasma membranes contain ab-type cytochrome that is rapidly reduced by ascorbic acid. The affinity towards ascorbate is 0.37 mM and is very similar to that of the chromaffin granule cytochromeb
561. High levels of cytochromeb reduction are reached when ascorbic acid is added either on the cytoplasmic or cell wall side of purified plasma membrane vesicles. This result points to a transmembrane organisation of the heme protein or alternatively indicates the presence of an effective ascorbate transport system. Plasma membrane vesicles loaded by ascorbic acid are capable of reducing extravesicular ferricyanide. Addition of ascorbate oxidase or washing of the vesicles does not eliminate this reaction, indicating the involvement of the intravesicular electron donor. Absorbance changes of the cytochromeb -band suggest the electron transfer is mediated by this redox component. Electron transport to ferricyanide also results in the generation of a membrane potential gradient as was demonstrated by using the charge-sensitive optical probe oxonol VI. Addition of ascorbate oxidase and ascorbate to the vesicles loaded with ascorbate results in the oxidation and subsequent re-reduction of the cytochromeb. It is therefore suggested that ascorbate free radical (AFR) could potentially act as an electron acceptor to the cytochrome-mediated electron transport reaction. A working model on the action of the cytochrome as an electron carrier between cytoplasmic and apoplastic ascorbate is discussed.Abbreviations AFR
ascorbate free radical
- AO
ascorbate oxidase
- DTT
dithiothreitol
- FCCP
carbonylcyanidep-trifluorome-thoxyphenylhydrazon
- Hepes
N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid)
- Oxonol
VI bis(3-propyl-5-oxoisoxazol-4-yl) penthamethine oxonol
- PMSF
phenylmethylsulfluoride 相似文献
11.
Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins that are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized and the putative tumor suppressor Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins is also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins. 相似文献
12.
Alajos Bérczi Filip Desmet Sabine Van Doorslaer Han Asard 《European biophysics journal : EBJ》2010,39(8):1129-1142
Tumor suppressor protein 101F6, a gene product of the 3p21.3 (human) and 9F1 (mouse) chromosomal region, has recently been
identified as a member of the cytochrome b561 (Cyt-b561) protein family by sequence homology. The His6-tagged recombinant mouse tumor suppressor Cyt-b561 protein (TSCytb) was recently expressed in yeast and purified, and the ascorbate reducibility was determined. TSCytb is
auto-oxidizable and has two distinct heme b centers with redox potentials of ~40 and ~140 mV. Its split α-band in the dithionite-reduced spectrum at both 295 and 77 K
is well resolved, and the separation between the two α-peaks is ~7 nm (~222 cm−1). Singular value decomposition analysis of the split α-band in the ascorbate-reduced spectra revealed the presence of two
major spectral components, each of them with split α-band but with different peak separations (6 and 8 nm). Similar minor
differences in peak separation were obtained when the split α-bands in ascorbate-reduced difference spectra at low (<1 mM)
and high (>10 mM) ascorbate concentrations were analysed. According to low-temperature electron paramagnetic resonance (EPR)
spectroscopy, the two heme b centers are in the low-spin ferric state with maximum principal g values of 3.61 and 2.96, respectively. These values differ from the ones observed for other members of the Cyt-b561 family. According to resonance Raman spectroscopy, the porphyrin rings are in a relaxed state. The spectroscopic results
are only partially in agreement with those obtained earlier for the native chromaffin granule Cyt-b561. 相似文献
13.
Bérczi A Su D Lakshminarasimhan M Vargas A Asard H 《Archives of biochemistry and biophysics》2005,443(1-2):82-92
Cytochromes b561 (Cyts b561) are ubiquitous membrane proteins catalyzing ascorbate-mediated trans-membrane electron transfer. A heterologous expression system in Saccharomyces cerevisiae was developed to study their structure-function relationship. Recombinant mouse chromaffin granule Cyt b561 (CGCytb) shows spectral characteristics, ascorbate reducibility, and redox potentials identical to that of the native bovine protein. Moreover, the reconstituted recombinant protein mediated trans-membrane electron transport with kinetic characteristics similar to that of bovine CGCytb. Site-directed mutant analysis supports the presence of two hemes coordinated by the highly conserved His pairs H52/H120 and H86/H159. Reduction of CGCytb by ascorbate showed biphasic kinetics (Kd1: 0.016 +/- 0.005 mM, Kd2: 1.24 +/- 0.19 mM). Mutation of a well-conserved Arg residue (R72) abolished high affinity CGCytb reduction by ascorbate, indicating that this residue may be critical for substrate binding. On the other hand, mutation of a Lys previously suggested to play a role in ascorbate binding (K83), did not affect the ascorbate-mediated reduction of the protein. 相似文献
14.
Summary The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb
561 (cytb
561). In this paper a rapid purification protocol for the cytb
561 of bean hypocotyls PM is described. An almost 200-fold increase of cytb
561 specific concentration was achieved with respect to the PM fraction, which contained about 0.2 nmol of ascorbate-reducible heme per mg protein. The procedure can be performed in one day starting from purified PMs obtained by the phase-partitioning procedure. However, cytb
561 proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cytb
561 eluted as a 130,000 Da protein-detergent complex from gel-filtration columns. It was completely reduced by ascorbate and reduced-minus-oxidized spectra showed -, - and -bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This work represents an initial approach to the biochemical characterization of the cytb
561 of higher plants, formerly suggested to be related to cytb
561 of animal chromaffin granules.Abbreviations cytb
561
cytochromeb
561
- PM
plasma membrane
- UPV
upper-phase vesicles
- GSII
glucan synthase II
- CCR
NADH-dependent cytochromec reductase
- CCO
cytochromec oxidase
- TX-100R
reduced Triton X-100 相似文献
15.
Cytochromes b561 (cyts b561) constitute a family of eukaryotic membrane proteins, catalysing ascorbate (Asc)-mediated trans-membrane electron transport, and hence likely involved in Asc regeneration. A class of proteins (DoH-CB) has been identified in plants and animals, containing the cyt b561 electron-transport domain (CB), combined with the catecholamine-binding regulatory domain of dopamine-beta-hydroxylase (DoH). A mammalian DoH-CB protein was previously reported to function as a cell-derived growth factor receptor (SDR2). We have performed an in silico analysis on DoH-CB proteins from Arabidopsis thaliana and demonstrate that structural features of both CB and DoH domains are well conserved. The combination of both domains may have evolved from a functional interaction between a cyt b561 and a DoH-containing protein, illustrating the so-called "Rosetta Stone" evolutionary principle, and this hypothesis is supported by sequence comparisons. DoH-CB proteins form a newly identified group of proteins, likely to play a key role in catecholamine action in plants. It is suggested that these proteins may function as trans-membrane electron shuttles, possibly regulated by catecholamines. The role and action of catecholamines in plants is poorly documented, but it is clear that they are involved in many aspects of growth and development. Whether the DoH-CB proteins functionally interact with Asc, as is the case for cyts b561, remains to be determined. 相似文献
16.
Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b561 family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at gmax = 3.7 corresponding to a highly anisotropic species, and another at gmax = 3.18; these species are similar to those observed in other cytochromes of the b561 family, and were reducible by ascorbate. In addition another signal was observed in some preparations at gmax = 2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of + 80 mV ± 30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b561 of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes. 相似文献
17.
(1) Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. (2) The midpoint redox potential of the cytochrome is 100–130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5–7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. (3) The Nernst plots of titration data have slopes of 75–115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. (4) Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. (5) Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4–6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis. 相似文献
18.
Summary The plasma membrane of higher plants contains more than one kind ofb-type cytochromes. One of these has a high redox potential and can be fully reduced by ascorbate. This component, the cytochromeb
561 (cytb
561), has its characteristic -band absorbance close to 561 nm wavelength at room temperature. Cytb
561 was first isolated from etiolated bean hook plasma membranes by two consecutive anion exchange chromatography steps. During the first step performed at pH 8, cytb
561 did not bind to the anion exchange column, but otherb-type cytochromes did. In the second step performed at pH 9.9, cytb
561 was bound to the column and was eluted from the column at an ionic strength of about 100 mM KCl. However, when the same protocol was applied to the solubilized plasma membrane proteins fromArabidopsis thaliana leaves and maize roots, the ascorbate-reducible cytb
561 bound already to the first anion exchange column at pH 8 and was eluted also at an ionic strength of about 100 mM KCl. Otherb-type cytochromes than the ascorbate-reducible cytb
561 from the plasma membranes of Arabidopsis leaves and maize roots showed similar Chromatographic characteristics to that of bean hypocotyls. These results demonstrate particular differences in the Chromatographic behavior of cytb
561 from different sources.Abbreviations cyt
b
561 cytochromeb
561
- PM
plasma membrane
- PAGE
polyacrylamide gel electrophoresis 相似文献
19.
Chloroplast fragments of a high fluorescent mutant of Chlamydomonas reinhardi, hfd 91, were compared against those of Acl+, a low chlorophyll variant of the wild type. The chloroplast fragments of the mutant which have a high invariant fluorescence yield lacked photochemical activities associated with photosystem II (PSII) but retained normal photosystem I (PSI) activities. The mutant fragments also lacked the low temperature (-196°C) light-induced absorbance changes due to the photoreduction of C-550 and the photooxidation of cytochrome (cyt) b-559 which are PSII-mediated reactions. A fourth-derivative analysis of the absolute spectra of the chloroplast fragments at different stages of reduction (obtained with ferricyanide, ascorbate, and dithionite) showed both the oxidized and reduced forms of C-550 and the reduced forms of cyt c-553, b-559, and b-564 in wild-type fragments. The mutant fragments lacked C-550 and an ascorbate-reducible cyt b-559 but contained cyt c-553, a dithionite-reducible cyt b-559, and cyt b-564. 相似文献
20.
Liu W Kamensky Y Kakkar R Foley E Kulmacz RJ Palmer G 《Protein expression and purification》2005,40(2):429-439
Bovine adrenal chromaffin granule cytochrome (cyt) b561 is a transmembrane hemoprotein that plays a key role in transporting reducing equivalents from ascorbate to dopamine-beta-hydroxylase for catecholamine synthesis. We have developed procedures for expression and purification of functional bovine adrenal cyt b561 in insect and yeast cell systems. The bovine cyt b561 coding sequence, with or without a hexahistidine-tag sequence at the C-terminus, was cloned into the pVL1392 transfer vector under the control of the polyhedrin promoter to generate recombinant baculovirus for protein expression in Sf9 insect cells (approximately 0.5 mg detergent-solubilized cyt b561/L culture). For the yeast system, the cyt b561 cDNA was modified with a hexahistidine-tag sequence at the C-terminus, and inserted into the pPICZB vector under the control of the alcohol oxidase promoter. The recombinant plasmid was transformed into Pichia pastoris GS115 competent cells to give methanol-inducible cyt b561 expression (approximately 0.7 mg detergent-solubilized cyt b561/L culture). Recombinant His-tagged cyt b561 expressed in Sf9 or Pichia cells was readily solubilized from membrane fractions with dodecyl maltoside and purified to electrophoretic homogeneity by one-step chromatography on Ni-NTA affinity resin. The purified recombinant cytochrome from both systems had a heme to protein ratio close to two and was fully functional, as judged by comparison with the spectroscopic and kinetic parameters of the endogenous cytochrome from chromaffin granules. A novel procedure for isolation of chromaffin granule membranes was developed to utilize frozen adrenal glands instead of fresh tissue. 相似文献