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91.
92.
93.
Weitz D Harder D Casagrande F Fotiadis D Obrdlik P Kelety B Daniel H 《The Journal of biological chemistry》2007,282(5):2832-2839
The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters. 相似文献
94.
Rabinovich ML Vasil'chenko LG Karapetyan KN Shumakovich GP Yershevich OP Ludwig R Haltrich D Hadar Y Kozlov YP Yaropolov AI 《Biotechnology journal》2007,2(5):546-558
Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine. 相似文献
95.
Kujawa M Volc J Halada P Sedmera P Divne C Sygmund C Leitner C Peterbauer C Haltrich D 《The FEBS journal》2007,274(3):879-894
We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K(m)). The enzyme may play a role in lignocellulose degradation. 相似文献
96.
Balijepalli RC Delisle BP Balijepalli SY Foell JD Slind JK Kamp TJ January CT 《Channels (Austin, Tex.)》2007,1(4):263-272
The localization of ion channels to specific membrane microdomains can impact the functional properties of channels and their role in cellular physiology. We determined the membrane localization of human Kv11.1 (hERG1) alpha-subunit protein, which underlies the rapidly activating, delayed rectifier K(+) current (I(Kr)) in the heart. Immunocytochemistry and membrane fractionation using discontinuous sucrose density gradients of adult canine ventricular tissue showed that Kv11.1 channel protein localized to both the cell surface and T-tubular sarcolemma. Furthermore, density gradient membrane fractionation using detergent (Triton X-100) and non-detergent (OptiPrep) methods from canine ventricular myocytes or HEK293 cells demonstrated that Kv11.1 protein, along with MiRP1 and Kv7.1 (KCNQ1) proteins, localize in cholesterol and sphingolipid enriched membrane fractions. In HEK293 cells, Kv11.1 channels, but not long QT-associated mutant G601S-Kv11.1 channels, also localized to cholesterol and sphingolipid enriched membrane fractions. Depletion of membrane cholesterol from HEK293 cells expressing Kv11.1 channels using methyl-beta-cyclodextrin (MbetaCD) caused a positive shift of the voltage dependence of activation and an acceleration of deactivation kinetics of Kv11.1 current (I(Kv11.1)). Cholesterol loading of HEK293 cells reduced the steep voltage dependence of I(Kv11.1) activation and accelerated the inactivation kinetics of I(Kv11.1). Incubation of neonatal mouse myocytes in MbetaCD also accelerated the deactivation kinetics of I(Kr). We conclude that Kv11.1 protein localizes in cholesterol and sphingolipid enriched membranes and that membrane cholesterol can modulate I(Kv11.1) and I(Kr). 相似文献
97.
The thermal denaturation of Lactobacillus confusus
l-2-Hydroxyisocaproate Dehydrogenase (l-HicDH) has been studied by Differential Scanning Calorimetry (DSC). The stability of this enzyme has been investigated at
different pH conditions. The results of this study indicate that the thermal denaturation of this enzyme is irreversible and
the T
m
is dependent on the scan-rate, which suggests that the denaturation process of l-HicDH is kinetically determined. The heat capacity function of l-HicDH shows a single peak with the T
m
values between 52.14°C and 55.89°C at pH 7.0 at different scan rates. These results indicate that the whole l-HicDH could unfold as a single cooperative unit, and intersubunit interactions of this homotetrameric enzyme must play a
significant role in the stabilization of the whole enzyme. The rate constant of the unfolding is analyzed as a first order
kinetic constant with the Arrhenius equation, and the activation energy has been calculated. The variation of the activation
energy values obtained with different methods does not support the validity of the one-step irreversible model. The denaturation
pathway was described by a three-state model, N → U → F, in which the dissociation of the tetramer takes place as an irreversible
step before the irreversible unfolding of the monomers. The calorimetric enthalpy associated with the irreversible dissociation
and the calorimetric enthalpy associated with the unfolding of the monomer were obtained from the best fitting procedure.
Thermal unfolding of l-HicDH was also studied using Circular Dichroism (CD) spectroscopy. Both methods yielded comparable values. 相似文献
98.
Distribution of haplotypes and microsatellite alleles among Asian elephants (<Emphasis Type="Italic">Elephas maximus</Emphasis>) in Thailand 总被引:1,自引:1,他引:0
Joerns Fickel Dietmar Lieckfeldt Parntep Ratanakorn Christian Pitra 《European Journal of Wildlife Research》2007,53(4):298-303
Habitat fragmentation often promotes increased inbreeding depression due to interrupted gene flow between populations. In
this study, we demonstrate that Asian elephants most likely also suffer from outbreeding depression due to cryptic speciation.
We analysed mitochondrial and nuclear DNA loci from 78 Asian elephants. Haplotype genealogy and analysis of molecular variance
revealed two matrilinear clades (α
h, β
h). Microsatellite analyses of individuals grouped according to their haplotype clade (corresponding group of nuclear genotypes
called α
nuc and β
nuc) revealed significant genotypic differentiation between α
nuc and β
nuc. Such genealogically differentiated forms in a morphologically uniform species are considered indicative of cryptic speciation.
The differentiation was caused by bulls, whereas considering cows only resulted in no differentiation. Such result is best
explained by Haldane’s rule whereby hybrid formation between genealogical forms causes lower viability and fertility of heterogametic hybrids. Although
the lack of hybrid-specific morphological characteristics renders direct testing of reduced hybrid fitness under natural conditions
unfeasible, the effects of Haldane’s rule are demonstrated by reduced male-mediated gene flow between genealogical forms under
sympatric conditions, as was indeed suggested by the data found in Asian elephants: male-mediated gene flow between groups
α
nuc and β
nuc was much lower than female-mediated gene flow.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
相似文献
Joerns FickelEmail: |
99.
Swan L. S. Sow Mark V. Brown Laurence J. Clarke Andrew Bissett Jodie van de Kamp Thomas W. Trull Eric J. Raes Justin R. Seymour Anna R. Bramucci Martin Ostrowski Philip W. Boyd Bruce E. Deagle Paula C. Pardo Bernadette M. Sloyan Levente Bodrossy 《Environmental microbiology》2022,24(5):2449-2466
We investigated the Southern Ocean (SO) prokaryote community structure via zero-radius operational taxonomic unit (zOTU) libraries generated from 16S rRNA gene sequencing of 223 full water column profiles. Samples reveal the prokaryote diversity trend between discrete water masses across multiple depths and latitudes in Indian (71–99°E, summer) and Pacific (170–174°W, autumn-winter) sectors of the SO. At higher taxonomic levels (phylum-family) we observed water masses to harbour distinct communities across both sectors, but observed sectorial variations at lower taxonomic levels (genus-zOTU) and relative abundance shifts for key taxa such as Flavobacteria, SAR324/Marinimicrobia, Nitrosopumilus and Nitrosopelagicus at both epi- and bathy-abyssopelagic water masses. Common surface bacteria were abundant in several deep-water masses and vice-versa suggesting connectivity between surface and deep-water microbial assemblages. Bacteria from same-sector Antarctic Bottom Water samples showed patchy, high beta-diversity which did not correlate well with measured environmental parameters or geographical distance. Unconventional depth distribution patterns were observed for key archaeal groups: Crenarchaeota was found across all depths in the water column and persistent high relative abundances of common epipelagic archaeon Nitrosopelagicus was observed in deep-water masses. Our findings reveal substantial regional variability of SO prokaryote assemblages that we argue should be considered in wide-scale SO ecosystem microbial modelling. 相似文献
100.
Dietmar Vestweber 《Journal of cellular biochemistry》1996,61(4):585-591
The selectins are carbohydrate-binding cell adhesion molecules acting in the vascular system. They mediate the docking of leukocytes to the blood vessel wall and the rolling of these cells along the endothelial cell surface. These adhesion phenomena initiate the entry of leukocytes into sites of inflammation as well as the migration of recirculating lymphocytes into secondary lymphoid tissues. Blocking selectin function with antibodies or oligosaccharides has proven to be beneficial in various animal models of inflammation and models of ischemia/reperfusion damage. This has raised much interest in the identification of the physiological ligands of the selectins. Several glycoprotein ligands have been identified, some of which can even be selectively isolated from cellular detergent extracts using a selectin as an affinity probe. Four of these “high affinity” ligands have been cloned. The structural requirements of their interaction with the selectins is discussed. © 1996 Wiley-Liss, Inc. 相似文献