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111.
112.
The glycogen synthase found in Pyrococcus furiosus is a hyperthermophilic biocatalyst that transfers the glucose portion of nucleotide-diphosphoglucose onto a growing carbohydrate biopolymer chain at 80 degrees C. In contrast to the mesophilic rabbit muscle glycogen synthase, the biocatalyst from P. furiosus possesses unusually broad nucleotide tolerance. The enzyme accepts all four common glucose-containing nucleotide-diphosphosugars: ADP-glucose, GDP-glucose, dTDP-glucose, and UDP-glucose. Using an electrospray ionization-mass spectroscopy (ESI-MS) assay, we determined the K(M) and Vmax for GDP-glucose to be 3.9 +/- 0.6 mM and 0.243 +/- 0.009 mM/min, and for dTDP-glucose to be 4.0 +/- 0.5 mM and 0.216 +/- 0.008 mM/min. A related nucleotide sugar, UDP-galactose, was not a reactive substrate, but was instead a competitive inhibitor with a Ki of 17 +/- 2 mM. The glycogen synthase from P. furiosus was shown not to have phosphorylase activity. The DeltaDeltaG of substrate binding was compared between the mesophilic rabbit muscle and the hyperthermophilic P. furiosus glycogen synthase to dissect any differences in sugar nucleotide recognition strategies at elevated temperatures. Both biocatalysts were shown to gain most of their substrate affinity through electrostatic interactions between the enzyme and the alpha-phosphate. 相似文献
113.
Kameritsch P Khandoga N Nagel W Hundhausen C Lidington D Pohl U 《Journal of cellular physiology》2005,203(1):233-242
Gap junction intercellular communication (GJIC) plays a significant role in the vascular system. Regulation of GJIC is a dynamic process, with alterations in connexin (Cx) protein expression and post-translational modification as contributing mechanisms. We hypothesized that the endothelial autacoid nitric oxide (NO) would reduce dye coupling in human umbilical vein endothelial cells (HUVECs). In our subsequent experiments, we sought to isolate the specific Cx isoform(s) targeted by NO or NO-activated signaling pathways. Since HUVEC cells variably express three Cx (Cx37, Cx40, and Cx43), this latter aim required the use of transfected HeLa cells (HeLaCx37, HeLaCx43), which do not express Cx proteins in their wild type form. Dye coupling was measured by injecting fluorescent dye (e.g., Alexa Fluor 488) into a single cell and determining the number of stained adjacent cells. Application of the NO donor SNAP (2 microM, 20 min) reduced dye coupling in HUVEC by 30%. In HeLa cells, SNAP did not reduce dye transfer of cells expressing Cx43, but decreased the dye transfer from Cx37-expressing cells to Cx43-expressing cells by 76%. The effect of SNAP on dye coupling was not mediated via cGMP. In contrast to its effect on dye coupling, SNAP had no effect on electrical coupling, measured by a double patch clamp in whole cell mode. Our results demonstrate that NO inhibits the intercellular transfer of small molecules by a specific influence on Cx37, suggesting a potential role of NO in controlling certain aspects of vascular GJIC. 相似文献
114.
Using transmission electron microscopy with glutaraldehyde and osmium tetroxide as chemical fixatives, hatshaped ascospores with two brims each were uncovered in the yeast Ambrosiozyma platypodis. This is the first report on such structures. 相似文献
115.
The glycolytic enzyme fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Catalysis of Schiff base forming class I FBPA relies on a number of intermediates covalently bound to the catalytic lysine. Using active site mutants of FBPA I from Thermoproteus tenax, we have solved the crystal structures of the enzyme covalently bound to the carbinolamine of the substrate fructose 1,6-bisphosphate and noncovalently bound to the cyclic form of the substrate. The structures, determined at a resolution of 1.9 A and refined to crystallographic R factors of 0.148 and 0.149, respectively, represent the first view of any FBPA I in these two stages of the reaction pathway and allow detailed analysis of the roles of active site residues in catalysis. The active site geometry of the Tyr146Phe FBPA variant with the carbinolamine intermediate supports the notion that in the archaeal FBPA I Tyr146 is the proton donor catalyzing the conversion between the carbinolamine and Schiff base. Our structural analysis furthermore indicates that Glu187 is the proton donor in the eukaryotic FBPA I, whereas an aspartic acid, conserved in all FBPA I enzymes, is in a perfect position to be the general base facilitating carbon-carbon cleavage. The crystal structure of the Trp144Glu, Tyr146Phe double-mutant substrate complex represents the first example where the cyclic form of beta-fructose 1,6-bisphosphate is noncovalently bound to FBPA I. The structure thus allows for the first time the catalytic mechanism of ring opening to be unraveled. 相似文献
116.
Reverse signaling through membrane-bound interleukin-15 总被引:5,自引:0,他引:5
Budagian V Bulanova E Orinska Z Pohl T Borden EC Silverman R Bulfone-Paus S 《The Journal of biological chemistry》2004,279(40):42192-42201
117.
Puchart V Vrsanská M Svoboda P Pohl J Ogel ZB Biely P 《Biochimica et biophysica acta》2004,1674(3):239-250
Two extracellular endo-beta-1,4-mannanases, MAN I (major form) and MAN II (minor form), were purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Molecular weights of MAN I and MAN II estimated by SDS-PAGE were 60 and 63 kDa, respectively. IEF afforded several glycoprotein bands with pI values in the range of 4.9-5.2 for MAN I and 4.75-4.9 for MAN II, each exhibiting enzyme activity. MAN I as well as MAN II showed highest activity at pH 4.5 and 60 degrees C and were stable in the pH range 4.5-8.5 and up to 55 degrees C. In accordance with the ability of the enzymes to catalyze transglycosylation reactions, 1H NMR spectroscopy of reaction products generated from mannopentaitol confirmed the retaining character of both enzymes. Both MAN I and MAN II exhibited essentially identical kinetic parameters for polysaccharides and a similar hydrolysis pattern of various oligomeric and polymeric substrates. Both beta-mannanases contained identical internal amino acid sequence corresponding to glycoside hydrolase family 5 and also a cellulose-binding module. These data suggested that both MAN I and MAN II are products of the same gene differing in posttranslational modification. Indeed, the corresponding gene was identified within the recently sequenced Aspergillus fumigatus genome (http://sanger.ac.uk/Projects/A_fumigatus/). 相似文献
118.
Behrmann I Smyczek T Heinrich PC Schmitz-Van de Leur H Komyod W Giese B Müller-Newen G Haan S Haan C 《The Journal of biological chemistry》2004,279(34):35486-35493
The Janus kinases are considered to be cytoplasmic kinases that constitutively associate with the cytoplasmic region of cytokine receptors, and the Janus kinases (Jaks) are crucial for cytokine signal transduction. We investigated Jak1 localization using subcellular fractionation techniques and fluorescence microscopy (immunofluorescence and yellow fluorescent protein-tagged Jaks). In the different experimental approaches we found Jak1 (as well as Jak2 and Tyk2) predominantly located at membranes. In contrast to previous reports we did not observe Jak proteins in significant amounts within the nucleus or in the cytoplasm. The cytoplasmic localization observed for the Jak1 mutant L80A/Y81A, which is unable to associate with cytokine receptors, indicates that Jak1 does not have a strong intrinsic membrane binding potential and that only receptor binding is crucial for the membrane recruitment. Finally we show that Jak1 remains a membrane-localized protein after cytokine stimulation. These data strongly support the hypothesis that cytokine receptor.Janus kinase complexes can be regarded as receptor tyrosine kinases. 相似文献
119.
Bareetseng AS Kock JL Pohl CH Pretorius EE Strauss CJ Botes PJ Van Wyk PW Nigam S 《Systematic and applied microbiology》2006,29(6):446-449
The distribution of 3-hydroxy oxylipins in Saturnispora saitoi was mapped using immunofluorescence microscopy. Fluorescence was observed on aggregating ascospores, indicating the presence of 3-hydroxy oxylipins on the surface or between ascospores. The oxylipin was identified as 3-hydroxy 9:1 using gas chromatography mass spectrometry. Furthermore, ultrastructural studies using scanning and transmission electron microscopy on ascospores revealed a clear equatorial ledge surrounding oval-shaped ascospores. 相似文献
120.
Francisco J. Enguita Ehmke Pohl David L. Turner Helena Santos Maria Arménia Carrondo 《Journal of biological inorganic chemistry》2006,11(2):189-196
The crystal structures of the oxidized and reduced forms of cytochrome c″ from Methylophilus methylotrophus were solved from X-ray synchrotron data to atomic resolution. The overall fold of the molecule in the two redox states is
very similar and is comparable to that of the oxygen-binding protein from the purple phototrophic bacterium Rhodobacter sphaeroides. However, significant modifications occur near the haem group, in particular the detachment from axial binding of His95 observed
upon reduction as well as the adoption of different conformations of some protonatable residues that form a possible proton
path from the haem pocket to the protein surface. These changes are associated with the previously well characterized redox-Bohr
behaviour of this protein. Furthermore they provide a model for one of the presently proposed mechanisms of proton translocation
in the much more complex protein cytochrome c oxidase. 相似文献