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1.
An X-ray structural study of human ceruloplasmin in relation to ferroxidase activity 总被引:5,自引:0,他引:5
P. F. Lindley Graeme Card Irina Zaitseva Vjacheslav Zaitsev Bengt Reinhammar Eva Selin-Lindgren Kunihiro Yoshida 《Journal of biological inorganic chemistry》1997,2(4):454-463
The role of ceruloplasmin as a ferroxidase in the blood, mediating the release of iron from cells and its subsequent incorporation into serum transferrin, has long been the subject of speculation and debate. However, a recent X-ray crystal structure determination of human ceruloplasmin at a resolution of around 3.0?Å, in conjunction with studies associating mutations in the ceruloplasmin gene with systemic haemosiderosis in humans, has added considerable weight to the argument in favour of a ferroxidase role for this enzyme. Further X-ray studies have now been undertaken involving the binding of the cations Co(II), Fe(II), Fe(III), and Cu(II) to ceruloplasmin. These results give insights into a mechanism for ferroxidase activity in ceruloplasmin. The residues and sites involved in ferroxidation are similar to those proposed for the heavy chains of human ferritin. The nature of the ferroxidase activity of human ceruloplasmin is described in terms of its three-dimensional molecular structure. 相似文献
2.
G. Musci Terri Z. L. Fraterrigo Lilia Calabrese David R. McMillin 《Journal of biological inorganic chemistry》1999,4(4):441-446
The possibility that ceruloplasmin (CP) functions as a copper transferase has fueled a continuing interest in studies of
the copper release process. The principal goal of the current investigation has been to identify the most labile copper centers
in sheep protein. In fact, subjecting the enzyme to a slow flux of cyanide at pH 5.2 under nitrogen in the presence of ascorbate
and a phenanthroline ligand produces partially demetalated forms of the protein. By standard chromatographic techniques it
is possible to isolate protein with a Cu/CP ratio of ∼4 or ∼5 as opposed to the native protein which has Cu/CP=5.8. In contrast
to other blue oxidases, analysis suggests that CP preferentially loses its type 1 coppers under these conditions. Thus, the
spectroscopic signals from the type 1 centers exhibit a loss of intensity while the EPR signal of the type 2 copper becomes
stronger. Furthermore, the Cu/CP≈4 and Cu/CP≈5 components retain about 50% of the activity of the native protein, consistent
with an intact type 2/type 3 cluster. All three type 1 copper sites appear to suffer copper loss. Reconstitution with a copper(I)
reagent restores the spectroscopic properties of the native protein and 90% of the original activity. The results suggest
a possible functional significance for the presence of three type 1 coppers in CP. By employing a pool of redox-active but
relatively labile type 1 copper centers, the enzyme can serve as a copper donor, if necessary, without completely sacrificing
its oxidase activity.
Received: 15 February 1999 / Accepted: 22 April 1999 相似文献
3.
M. M. Nielsen E. S. Seo A. Dilokpimol J. Andersen M. Abou Hachem H. Naested 《Biocatalysis and Biotransformation》2013,31(1-2):59-67
Germinating barley seeds contain multiple forms of α-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The α-amylases are endo-acting and possess a long substrate binding cleft with a characteristic subsite binding energy profile around the catalytic site. Furthermore, several amylolytic enzymes that facilitate attack on the natural substrate, i.e. the endosperm starch granules, have secondary sugar binding sites either situated on the surface of the protein domain or structural unit that contains the catalytic site or belonging to a separate starch binding domain. The role of surface sites in the function of barley α-amylase 1 has been investigated by using mutational analysis in conjunction with carbohydrate binding analyses and crystallography. The ability to bind starch depends on the surface sites and varies for starch granules of different genotypes and botanical origin. The surface sites, moreover, are candidates for being involved in degradation of polysaccharides by a multiple attack mechanism. Future studies of the molecular nature of the multivalent enzyme-substrate interactions will address surface sites in both barley α-amylase 1 and in the related isozyme 2. 相似文献
4.
The trace element contamination levels in mollusks were evaluated for different marine coastal sites in the Mediterranean
(Israeli coast), Red (Israeli coast) and North (German coast) Seas. Three bivalve species (Mactra corallina, Donax sp, and Mytilus
edulis) and two gastropod species (Patella sp.and Cellana rota) were sampled at polluted and relatively clean sites, and their soft tissue analyzed for Hg, Cd, Zn, Cu, Mn and Fe concentrations.
Representative samples were screened for organic contaminants [(DDE), polychlorinated biphenyls PCBs and polycyclic aromatic
hydrocarbons (PAHs)] which exhibited very low concentrations at all sites. In the Red Sea, the gastropod C. rota showed low levels of Hg (below detection limit) and similar Cd concentrations at all the examined sites, while other trace
elements (Cu, Zn, Mn, Fe) were slightly enriched at the northern beach stations. Along the Mediterranean coast of Israel,
Hg and Zn were enriched in two bivalves (M. corallina and Donax sp.) from Haifa Bay, both species undergoing a long-term decrease in Hg based on previous studies. Significant Cd and Zn enrichment
was detected in Patella sp. from the Kishon River estuary at the southern part of Haifa Bay. In general, Patella sp. and Donax sp. specimens from Haifa Bay exhibited higher levels of Cd compared to other sites along the Israeli Mediterranean coast,
attributed to the enrichment of Cd in suspended particulate matter. Along the German coast (North Sea) M. edulis exhibited higher concentrations of Hg and Cd at the Elbe and Eider estuaries, but with levels below those found in polluted
sites elsewhere.
Received: 25 February 1999 / Received in revised form: 22 April 1999 / Accepted: 30 April 1999 相似文献
5.
Tight linkage may cause a reduction of nucleotide diversity in a chromosomal region if an advantageous mutation appears in
that region which is driven to fixation by directional selection. This process is usually called genetic hitchhiking. If selection
is strong, the entire process takes place during a time period of length 2s ln (2N) that is very short relative to 2N generations [s is the selection coefficient of the advantageous mutation and N the effective diploid population size]. On the time scale of 2N generations, which is characteristic for neutral evolution, we may therefore call this process a hitchhiking event. Using
coalescent methods, we analyzed a model in which a hitchhiking event occurred in a chromosomal region of zero-recombination
in the past at time x. Such a hitchhiking “catastrophe” wipes out completely genetic variation that existed in a population before that time. Standing
variation observed at present must therefore be due to mutations that have arisen since time point x. Assuming that all newly arising mutations are neutral, we derived expressions for the expectation, variance and also for
the higher moments of the number of nucleotide sites segregating in a sample of n genes as a function of x. The result for the first moment is then used to estimate the time back to the last hitchhiking event based on DNA polymorphism
data from Drosophila. Assuming that directional selection is the sole determinant of the level of genetic variation in the gene regions surveyed,
we obtained estimates of x that were typically in the order of 0.1N generations.
Received 14 May 1996; received in revised form 26 August 1996 相似文献
6.
Protein- and pH-dependent binding of nascent pectin and glucuronoarabinoxylan to xyloglucan in pea 总被引:4,自引:0,他引:4
Nascent pectin and glucuronoarabinoxylan, synthesised in vitro by membrane-bound enzymes from etiolated pea (Pisum sativum L.) epicotyls, were found to bind to pea xyloglucan in a pH-dependent manner. The binding was maximum at low pH (3–4), and
decreased to almost zero at pH 6. The binding was probably non-covalent and reached saturation within 5 min. Removal of the
fucose residues of xyloglucan decreased the degree of binding. Removal by protease of the proteins attached to nascent pectin
and glucuronoarabinoxylan greatly reduced the maximum binding and abolished the pH-dependence. The observed binding may be
of considerable significance in the process of cell-wall assembly and in the control of cell extension.
Received: 4 November 1999 / 22 December 1999 相似文献
7.
Interactions of azide ion with bovine heart cytochrome c oxidase (CcO) at five redox levels (IV) to (0), obtained by zero to four electron reduction of fully oxidized enzyme CcO(IV), were monitored by infrared and visible/Soret spectra. Partially reduced CcO gave three azide asymmetric stretch band at 2040, 2016, and 2004 cm-1 for CcO(III)N3 and two at 2040 and 2016 cm-1 for CcO(II)N3 and CcO(I)N3. Resting CcO(IV) reacts with N3- to give one band at 2041 cm-1 assigned to CuB2+N3 and another at 2051 cm-1 to N3- that is associated with protein but is not bound to a metal ion. At high azide concentrations the weak association of many azide molecules with non-metal protein sites was observed at all redox levels. These findings provide direct evidence for 1) N3- binding to CuB as well as Fea3 in partially reduced enzyme, but no binding to Fea3 in fully oxidized enzyme and no binding to either metal in fully reduced enzyme; 2) a long range effect of the oxidation state of Fea or CuA on ligand binding at heme a3, but not at CuB; and 3) an insensitivity of either Fea3 or CuB ligand site to changes in ligand or oxidation state at the other site. The observed independence of the Fea3 and CuB sites provides further support for Fea3(3)+ OOH, rather than Fea3(3)+ OOCuB2+, as an intermediate in the reduction of O2 to water by the oxidase. 相似文献
8.
Polymorphism, distribution, and segregation of AFLP markers in a doubled haploid rice population 总被引:18,自引:0,他引:18
M. Maheswaran P. K. Subudhi S. Nandi J. C. Xu A. Parco D. C. Yang N. Huang 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,94(1):39-45
We exploited the newly developed amplified fragment length polymorphism (AFLP) technique to study the polymorphism, distribution
and inheritance of AFLP markers with a doubled haploid rice population derived from ‘IR64’/‘Azucena’. Using only 20 pairs
of primer combinations, we detected 945 AFLP bands of which 208 were polymorphic. All 208 AFLP markers were mapped and distributed
over all 12 chromosomes. When these were compared with RFLP markers already mapped in the population, we found the AFLP markers
to be highly polymorphic in rice and to follow Mendelian segregation. As linkage map of rice can be generated rapidly with
AFLP markers they will be very useful for marker-assisted backcrossing.
Received: 11 April 1996 / Accepted: 14 June 1996 相似文献
9.
Soybean Kunitz, C-II and PI-IV inhibitor genes confer different levels of insect resistance to tobacco and potato transgenic plants 总被引:1,自引:0,他引:1
S. Marchetti M. Delledonne C. Fogher C. Chiabà F. Chiesa F. Savazzini A. Giordano 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,101(4):519-526
In modern, highly intensive agriculture, the control of insect pests is basically achieved with the application of chemical
pesticides. Heavy reliance on this sole strategy is associated with several drawbacks, and the development of alternative
or complementary methods to chemical control is desirable. In this work, three soybean genes (KTi
3
, C-II and PI-IV)coding for serine proteinase inhibitors were isolated by PCR and transferred to Agrobacterium tumefaciens EHA 105, which in turn was used for transforming tobacco leaf and potato tuber discs. Biochemical assays confirmed that transgenic
plants synthesized serine proteinase inhibitors; rates of expression varied among plants. The level of insect resistance (tested
with Spodoptera littoralis Boisduval) was particularly high in tobacco, where many plants caused the death of all larvae. In potatoes, larval mortality
was much less frequently achieved, but the results were still encouraging in that larval weight gain was reduced by 50% in
the presence of adequate amounts of inhibitor. When 8-day-old larvae were fed different KTi
3
-expressing tobacco plants, a highly significant (P<0.01) correlation was observed between inhibitor content and larval live weight. Larval weight gain was found to be dependent
on midgut proteolytic activity. On the basis of the evidence collected, it is suggested that further work is required to identify
more specific inhibitors for the main proteinases of the target insect.
Received: 30 March 1998 / Accepted: 9 December 1999 相似文献
10.
Influence of organic amendments on arbuscular mycorrhizal fungi in relation to rice sheath blight disease 总被引:2,自引:0,他引:2
The effect of various organic soil amendments on arbuscular myorrhizal (AM) fungal activity on rice plants was tested under
greenhouse and field conditions with reference to sheath blight (ShB) disease caused by Rhizoctonia solani. AM spore density, per cent infection, and intensity of infection were increased by organic amendments, whilst ShB disease
was decreased. Certain amendments, especially green leaf manure, stimulated arbuscule development in rice plants. Mycorrhiza
formation and sporulation were higher with healthy rice plants than with rice plants infected with R. solani. Our results indicate the possibility of using selective organic amendments to enhance development of native AM fungi and
thus reduce disease incidence.
Accepted: 9 November 1995 相似文献
11.
Colin G. Saysell Terez Barna Christopher D. Borman Andrew J. Baron Michael J. McPherson A. G. Sykes 《Journal of biological inorganic chemistry》1997,2(6):702-709
The indole ring of Trp-290 in galactose oxidase has an important role in restricting entry to the substrate-binding (Cu) site of galactose oxidase via a short ~8?Å access pocket/channel. It also overlays and helps stabilise the radical-forming Cu-coordinated Tyr-272, reduction potential 400?mV. In this paper the effect of replacing Trp-290 by the less bulky His residue is explored at 25??°C, I=0.100?M (NaCl), and different effects are quantified. Interactions with buffers, not observed in the case of wild-type (WT) GOase, have been investigated by UV-Vis spectrophotometry on the non-radical GOasesemi (CuII) form of the Trp290His variant. Equilibrium constants K eq/M–1 from absorbance changes at 635?nm are for 1?:?1 interactions with the OH-containing buffers H2PO4 – (231), Hepes (43) and Tris (202), concentrations 0–60?mM. No similar interactions are observed with Mes, Lutidine and Ches, when significantly different UV-Vis spectra with no peak at ~635?nm are obtained. At pH 7.5 the reduction potential for the Trp290His GOaseox/GOasesemi couple is 730?mV, which compares with 400?mV for the WT GOase couple. Consistent with the 730?mV value the GOasesemi form is not oxidised with [Fe(CN)6]3– (410?mV) or [W(CN)8]3– (530?mV), and much stronger oxidants such as [Mo(CN)8]3– (800?mV) and [IrCl6]2– (890?mV) are required. The GOaseox product is unstable and decays within 20?min with re-formation of GOasesemi. From changes in UV-Vis spectra with pH, Trp290His GOasesemi gives a pK a of 6.9, and rate constants for the oxidation of GOasesemi with [Mo(CN)8]3– are dependent on this same pK a. The latter compares with 7.9 for WT GOasesemi, and is assigned here also as protonation of Tyr-495. The 1?:?1 binding of azide at the substrate-binding (H2O) site of Trp290His GOasesemi was studied and gives a formation constant 330?M–1 at pH 7.5, which is an order of magnitude less than the corresponding value for WT GOasesemi. The trends observed indicate less affinity of Trp290His GOasesemi for the ionic reactants H+ and N3 –. 相似文献
12.
A. D. Blest Sally Stowe 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1997,180(4):347-355
(1) In vitro retinas of a crab, Leptograpsus, were treated with a phospholipase inhibitor, manoalide, or a G-protein activator, Mas-7. Both drugs address early stages
of the phototransduction cascade. (2) Manoalide inhibited the light-dependent reduction of rhabdoms during the `day' phase
of the light cycle, but did not induce rhabdom overgrowth. Following a period of darkness manoalide failed to affect the diminution
of illuminated rhabdoms. (3) The diminution of rhabdoms that follows photoreceptor depolarisation induced by 100 mmol · l−1 K+ in darkness was not affected by 2␣μmol · l−1 manoalide. (4) When retinas in the `night' phase were treated with Mas-7 in darkness, rhabdom diameters were augmented, concurrently
with endocytosis of photoreceptor plasma membranes. (5) The results of combining manoalide and Mas-7 with actinomycin D, U-57908
or okadaic acid, drugs used in previous studies to manipulate steps notionally lower in the transduction cascade, lead to
a hypothetical model for the regulation of phototransductive membrane turnover by arthropods.
Accepted: 3 October 1996 相似文献
13.
14.
15.
Different types of nursery inocula formulations, namely mixed indigenous cultures and Glomus intraradices Schenck and Smith, were compared with commercially available inoculants of AM fungi in a pot experiment using two horticultural
crops, Capsicum and Polianthes. Soil-based inocula and soil beads produced the highest response in both crops. Glomus intraradices resulted in the highest yield in both Polianthes (45% increase in spike length) and Capsicum (112% increase in fruit yield). Among the commercial inocula tested, only Mycorise enhanced spike length (33%) and fruit
yield (11%) in the two hosts. Overall AM colonization was higher in Polianthes than in Capsicum. Sheared root inocula of G. intraradices resulted in high colonization (upto 68%) but the yield enhancement was lower than with soil-based formulations. The mixed
indigenous culture produced the highest number of spores and propagules and commercial inocula the lowest.
Accepted: 2 February 1998 相似文献
16.
Christopher D. Borman Colin G. Saysell A. G. Sykes 《Journal of biological inorganic chemistry》1997,2(4):480-487
Reactions (25 °C) of galactose oxidase, GOaseox from Fusarium NRRL 2903 with five different primary-alcohol-containing substrates RCH2OH:- D-galactose (I) and 2-deoxy-d-galactose (II) (monosaccharides); methyl-β-d-galactopyranoside (III) (glycoside);d-raffinose (IV) (trisaccharide); and dihydroxyacetone (V) have been studied in the presence of O2. The GOaseox state has a tyrosyl radical coordinated at a square-pyramidal CuII active site, and is a two-equivalent oxidant. Reactant concentrations were [GOaseox] (0.8–10 μM), RCH2OH (1.0–6.0 mM), and O2 (0.14–0.29 mM), with I=0.100 M (NaCl). The reactions, monitored at 450 nm by stopped-flow spectrophotometry, terminated with depletion of the O2. Each trace was fitted to the competing reactions GOaseox+RCH2 OH → GOaseredH2+RCHO (k
1), and GOaseredH2+O2→ GOaseox+H2O2 (k
2), with GOaseredH2 written as the doubly protonated two-electron-reduced CuI product. It was necessary to avoid auto-redox interconversion of GOaseox and GOasesemi . Information obtained at pH 7.5 indicates a 5 : 95 (ox : semi) "native" mix equilibration complete in ∼3 h. At pH >7.5,
rate constants 10–4 k
1 / M–1 s–1 for the reactions of GOaseox with (I) (1.19), (II) (1.07), (III) (1.29), (IV) (1.81), (V) (2.94) were determined. On decreasing the pH to 5.5, k
1 values decreased by factors of up to a half, and acid dissociation pK
as in the range 6.6–6.9 were obtained. UV-Vis spectrophotometric studies on GOaseox gave an independently determined pK
a of 6.7. No corresponding reactions of the Tyr495Phe variant were observed, and there are no similar UV-Vis absorbance changes
for this variant. The pK
a is therefore assigned to protonation of Tyr-495 which is a ligand to the Cu. The rate constant k
2 (1.01×107 M–1 s–1) is independent of pH in the range 5.5–9.0 investigated, suggesting that H+ (or H-atoms) for the O2 → H2O2 change are provided by the active site of GOasered . The CuI of GOasered is less extensively complexed, and a coordination number of three is likely.
Received: 4 February 1997 / Accepted: 16 May 1997 相似文献
17.
Cheng CM Lee YJ Wang WT Hsu CT Tsai JS Wu CM Ou KL Yang TS 《Biochemical and biophysical research communications》2011,(1):297-301
Accurately predicting binding affinity constant (KA) is highly required to determine the binding energetics of the driving forces in drug–DNA interactions. Recently, PD153035, brominated anilinoquinazoline, has been reported to be not only a highly selective inhibitor of epidermal growth factor receptor but also a DNA intercalator. Here, we use a dual-trap optical tweezers to determining KA for PD153035, where KA is determined from the changes in B-form contour length (L) of PD153035–DNA complex. Here, L is fitted using a modified wormlike chain model. We found that a noticeable increment in L in 1 mM sodium cacodylate was exhibited. Furthermore, our results showed that KA = 1.18(±0.09) × 104 (1/M) at 23 ± 0.5 °C and the minimum distance between adjacent bound PD153035 ≈ 11 bp. We anticipate that by using this approach we can determine the complete thermodynamic profiles due to the presence of DNA intercalators. 相似文献
18.
Frelet-Barrand A Kolukisaoglu HU Plaza S Rüffer M Azevedo L Hörtensteiner S Marinova K Weder B Schulz B Klein M 《Plant & cell physiology》2008,49(4):557-569
The enormous metabolic plasticity of plants allows detoxificationof many harmful compounds that are generated during biosyntheticprocesses or are present as biotic or abiotic toxins in theirenvironment. Derivatives of toxic compounds such as glutathioneconjugates are moved into the central vacuole via ATP-bindingcassette (ABC)-type transporters of the multidrug resistance-associatedprotein (MRP) subfamily. The Arabidopsis genome contains 15AtMRP isogenes, four of which (AtMRP1, 2, 11 and 12) clustertogether in one of two major phylogenetic clades. We isolatedT-DNA knockout alleles in all four highly homologous AtMRP genesof this clade and subjected them to physiological analysis toassess the function of each AtMRP of this group. None of thesingle atmrp mutants displayed visible phenotypes under controlconditions. In spite of the fact that AtMRP1 and AtMRP2 hadbeen described as efficient ATP-dependent organic anion transportersin heterologous expression experiments, the contribution ofthree of the AtMRP genes (1, 11 and 12) to detoxification ismarginal. Only knockouts in AtMRP2 exhibited a reduced sensitivitytowards 1-chloro-2,4-dinitrobenzene, but not towards other herbicides.AtMRP2 but not AtMRP1, 11 and 12 is involved in chlorophylldegradation since ethylene-treated rosettes of atmrp2 showedreduced senescence, and AtMRP2 expression is induced duringsenescence. This suggests that AtMRP2 is involved in vacuolartransport of chlorophyll catabolites. Vacuolar uptake studiesdemonstrated that transport of typical MRP substrates was reducedin atmrp2. We conclude that within clade I, only AtMRP2 contributessignificantly to overall organic anion pump activity in vivo. 相似文献
19.
An increasing number of genes are known to show expression in the cranial neural crest area. So far it is very difficult to
analyze their effect on neural crest cell migration because of the lack of transplantation techniques. This paper presents
a simple method to study the migratory behavior of cranial neural crest cells by homo- and heterotopic transplantations: Green
fluorescent protein (GFP) RNA was injected into one blastomere of Xenopus laevis embryos at the 2-cell stage. The cranial neural crest area of stage 14 embryos was transplanted into the head or trunk region
of an uninjected host embryo, and the migration was monitored by GFP fluorescence. The transplants were further examined by
double immunostaining and confocal microscopy to trace migratory routes inside the embryo, and to exclude contaminations of
grafts with foreign tissues. Our results demonstrate that we developed a highly efficient and reproducible technique to study
the migratory ability of cranial neural crest cells. It offers the possibility to analyze genes involved in neural crest cell
migration by coinjecting their RNA with that of GFP.
Received: 28 September 1999 / Accepted: 17 November 1999 相似文献
20.
R. E. Voorrips M. C. Jongerius H. J. Kanne 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,94(1):75-82
A genetic map covering 615 cM in 12 linkage groups was assembled based on 92 RFLP and AFLP markers segregating in a population
of 107 doubled haploid lines (DH lines) of Brassica oleracea. The DH-line population was obtained through microspore culture from the of two homozygous parents: DH-line Bi derived from the cabbage landrace Bindsachsener, and DH-line Gr from broccoli cv ‘Greenia’.
Sixty-five percent of the loci, and in some cases complete linkage groups, displayed distorted segregation ratios, a frequency
much higher than that observed in populations of the same species. DH-line Bi was resistant to clubroot, which is caused by a Dutch field isolate of Plasmodiophora brassicae. Resistance in the DH-line population was determined in two ways: by assigning symptom grades to each plant, and by measuring
the fresh weights of the healthy and affected parts of the root system of each plant. Using a multiple QTL mapping approach
to analyze the fresh weight data, we found two loci for clubroot resistance; these were designated pb-3 and pb-4. The additive effects of these loci were responsible for 68% of the difference between the parents and for 60% of the genetic
variance among DH-line means. Also, indications for the presence of two additional, minor QTLs were found. Analysis of symptom
grades revealed the two QTLs pb-3 and pb-4, as well as one of the two minor QTLs indicated by analysis of the fresh weight data.
Received: 29 April 1996 / Accepted: 10 May 1996 相似文献