Each of the two beta peptides which comprise the B2 protein of Escherichia coli ribonucleotide reductase (RRB2) possesses a nonheme dinuclear iron cluster and a tyrosine residue at position 122. The oxidized form of the protein contains all high spin ferric iron and 1.0-1.4 tyrosyl radicals per RRB2 protein. In order to define the stoichiometry of in vitro dioxygen reduction catalyzed by fully reduced RRB2 we have quantified the reactants and products in the aerobic addition of Fe(II) to metal-free RRB2apo utilizing an oxygraph to quantify oxygen consumption, electron paramagnetic resonance to measure tyrosine radical generation, and M?ssbauer spectroscopy to determine the extent of iron oxidation. Our data indicate that 3.1 Fe(II) and 0.8 Tyr122 are oxidized per mol of O2 reduced. M?ssbauer experiments indicate that less than 8% of the iron is bound as mononuclear high spin Fe(III). Further, the aerobic addition of substoichiometric amounts of 57Fe to RRB2apo consistently produces dinuclear clusters, rather than mononuclear Fe(III) species, providing the first direct spectroscopic evidence for the preferential formation of the dinuclear units at the active site. These stoichiometry studies were extended to include the phenylalanine mutant protein (Y122F)RRB2 and show that 3.9 mol-equivalents of Fe(II) are oxidized per mol of O2 consumed. Our stoichiometry data has led us to propose a model for dioxygen activation catalyzed by RRB2 which invokes electron transfer between iron clusters. 相似文献
X-ray crystallographic studies of the intradiol cleaving protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have shown that the enzyme has a trigonal bipyramidal ferric active site with two histidines, two tyrosines, and a solvent molecule as ligands [Ohlendorf, D.H., Lipscomb, J.D., & Weber, P.C. (1988) Nature 336, 403-405]. Fe K-edge EXAFS studies of the spectroscopically similar protocatechuate 3,4-dioxygenase from Brevibacterium fuscum are consistent with a pentacoordinate geometry of the iron active site with 3 O/N ligands at 1.90 A and 2 O/N ligands at 2.08 A. The 2.08-A bonds are assigned to the two histidines, while the 1.90-A bonds are associated with the two tyrosines and the coordinated solvent. The short Fe-O distance for the solvent suggests that it coordinates as hydroxide rather than water. When the inhibitor terephthalate is bound to the enzyme, the XANES data indicate that the ferric site becomes 6-coordinate and the EXAFS data show a beat pattern which can only be simulated with an additional Fe-O/N interaction at 2.46 A. Together, the data suggest that the oxygens of the carboxylate group in terephthalate displace the hydroxide and chelate to the ferric site but in an asymmetric fashion. In contrast, protocatechuate 3,4-dioxygenase remains 5-coordinate upon the addition of the slow substrate homoprotocatechuic acid (HPCA). Previous EPR data have indicated that HPCA forms an iron chelate via the two hydroxyl functions.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
TRPA1 and TRPV1 channels respond to external stimulation as pain mediators and form a complex with a transmembrane protein TMEM100 in some tissues. However, their expression and interaction in dental pulp is unclear. To investigate the functional co-expression of TRPA1 channel, TRPV1 channel and TMEM100 in human odontoblasts (HODs), immunohistochemistry, immunofluorescence staining and Western blot were used to study their co-localization and expression in both native HODs and cultured HOD-like cells. Calcium imaging was used to detect the functional interaction between TRPA1 and TRPV1 channels. Immunohistochemistry and multiple immunofluorescence staining of tooth slices showed positive expression of TRPA1 channel, TRPV1 channel and TMEM100 mainly in the cell bodies of HODs, and TRPA1 channel presented more obvious immunofluorescence in the cell processes than TRPV1 channel and TMEM100. HALO software analysis showed that TRPA1 and TRPV1 channels were positively expressed in most TMEM100+ HODs and these three proteins were strongly correlated in HODs (P < 0.01). The protein expression levels of TRPA1 channel, TRPV1 channel and TMEM100 in HODs showed no significant difference (P?>?0.05). Double immunofluorescence staining of cultured HOD-like cells visually demonstrated that TRPA1 and TRPV1 channel were both highly co-localized with TMEM100 with similar expressive intensity. Calcium imaging showed that there was a functional interaction between TRPA1 and TRPV1 channels in HOD-like cells, and TRPA1 channel might play a greater role in this interaction. Overall, we concluded that TRPA1 channel, TRPV1 channel and TMEM100 could be functionally co-expressed in HODs.
AbstractHyperglycaemia is associated with the poor outcome after intracerebral haemorrhage (ICH). Acetazolamide (AZA), a kind of carbonic anhydrogenase (CA) inhibitor, its effectiveness in ICH had been reported. However, the connections between AZA and ICH, especially in hyperglycaemia condition had never been defined. In this study, adult Sprague–Dawley rats were administered with vehicle or streptozotocin (STZ) to render them into normoglycaemic (NG) or hyperglycaemic (HG), respectively. Collagenase was then injected into the striatum. The NG or HG ICH rats treated with vehicle control or 5?mg/kg AZA (oral gavage) underwent haemorrhagic area assessments on the 1st, 4th, and 7th day after ICH. The coverage of pericytes was examined by immunohistochemistry. Reactive oxygen species (ROS) levels were assessed in mouse astrocyte cell line treated with vehicle or 20?μmol/L of AZA in culture media according to two different glucose concentrations. AZA reduced the haematoma size, improved neurobehavioral functions, suppressed astrocytic ROS production in vitro, and preserved cerebral pericytes coverage, which are even more remarkable in HG conditions. The present study indicates that AZA may alleviate some sequelae after ICH, especially in poorer prognostic HG rats through the suppression of astrocytic ROS production. 相似文献
The Pacific oyster (Crassostrea gigas) is globally distributed and is one of the most commercially and ecologically important marine organisms. However, little is known about the genome of this species. In this study, a C. gigas fosmid library was constructed that contains 459,936 clones with an average insert size of approximately 40 kb, representing 22.34-fold haploid genome equivalents. End sequencing generated 90,240 fosmid end sequences (FESs) with an average length of 384.27 base pairs (bp), covering approximately 2.58% of the Pacific oyster genome. The FESs were subsequently assembled and annotated, resulting in 6332 sequences with predicted open reading frames≥300 and 1,189,100 bp repeats. Furthermore, a total of 3200 microsatellite repeats were identified, and dinucleotide repeats were found to occur most abundantly, with AG and AAT being the most abundant repeat class of dinucleotides and trinucleotides. We also found that the repeat number was generally negatively proportional to the repeat element length. Microsatellites composition between the transcribed sequences and genomic sequences was shown to be different. Point mutations of microsatellite were non-random and underwent strong selection stress. Overall, a comprehensive sequence resource for the Pacific oyster was created, including annotated transposable elements, tandem repeats, protein coding sequences and microsatellites. These initial findings will serve as resources for further in-depth studies of physical mapping, gene discovery, microsatellite marker developing and evolution studies. 相似文献