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91.
Proexosite-1 on prothrombin is a factor Va-dependent recognition site for the prothrombinase complex
Although the contribution of basic residues of exosite-1 to the catalytic function of thrombin has been studied extensively, their role in the specificity of prothrombin recognition by factor Xa in the prothrombinase complex (factor Xa, factor Va, phosphatidylcholine/phosphatidylserine vesicles, and Ca2+) has not been examined. In this study, we prepared several mutants of prethrombin-1 (prothrombin lacking Gla and Kringle-1 domains) in which basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77 in chymotrypsinogen numbering) were individually substituted with a Glu. Following expression in mammalian cells and purification to homogeneity, these mutants were characterized with respect to their ability to function as zymogens for both factor Xa and the prothrombinase complex. Factor Xa by itself exhibited similar catalytic activity toward both the wild type and mutant substrates; however, its activity in the prothrombinase complex toward most of mutants was severely impaired. Further kinetic studies in the presence of Tyr63-sulfated hirudin-(54-65) peptide suggested that although the peptide inhibits the prothrombinase activation of the wild type zymogen with a KD of 0.5-0.7 microm, it is ineffective in inhibiting the activation of mutant zymogens (KD = 2-30 microm). These results suggest that basic residues of proexosite-1 on prothrombin are factor Va-dependent recognition sites for factor Xa in the prothrombinase complex. 相似文献
92.
Thrombomodulin (TM) functions as a cofactor to enhance the rate of protein C activation by thrombin approximately 1000-fold. The molecular mechanism by which TM improves the catalytic efficiency of thrombin toward protein C is not known. Molecular modeling of the protein C activation based on the crystal structure of thrombin in complex with the epidermal growth factor-like domains 4, 5, and 6 of TM (TM456) predicts that the binding of TM56 to exosite 1 of thrombin positions TM4 so that a negatively charged region on this domain juxtaposes a positively charged region of protein C. It has been hypothesized that electrostatic interactions between these oppositely charged residues of TM4 and protein C facilitate a proper docking of the substrate into the catalytic pocket of thrombin. To test this hypothesis, we have constructed several mutants of TM456 and protein C in which charges of the putative interacting residues on both TM4 (Asp/Glu) and protein C (Lys/Arg) have been reversed. Results of TM-dependent protein C activation studies by such a compensatory mutagenesis approach support the molecular model that TM4 interacts with the basic exosite of protein C. 相似文献
93.
FRET (fluorescence resonance energy transfer) studies have shown that the vitamin K-dependent coagulation proteases bind to membrane surfaces perpendicularly, positioning their active sites above the membrane surfaces. To investigate whether EGF (epidermal growth factor) domains of these proteases play a spacer function in this model of the membrane interaction, we used FRET to measure the distance between the donor fluorescein dye in the active sites of Fl-FPR (fluorescein-D-Phe-Pro-Arg-chloromethane)-inhibited fXa (activated Factor Xa) and its N-terminal EGF deletion mutant (fXa-desEGF1), and the acceptor OR (octadecylrhodamine) dye incorporated into phospholipid vesicles composed of 80% phosphatidylcholine and 20% phosphatidylserine. The average distance of closest approach (L) between fluorescein in the active site and OR at the vesicle surface was determined to be 56+/-1 A (1 A=0.1 nm) and 63+/-1 A for fXa-desEGF1 compared with 72+/-2 A and 75+/-1 A for fXa, in the absence and presence of fVa (activated Factor V) respectively, assuming kappa2=2/3. In comparison, an L value of 95+/-6 A was obtained for a S195C mutant of fXa in the absence of fVa in which fluorescein was attached directly to Cys(195) of fXa. These results suggest that (i) EGF1 plays a spacer function in holding the active site of fXa above the membrane surface, (ii) the average distance between fluorescein attached to Fl-FPR in the active site of fXa and OR at the vesicle surface may not reflect the actual distance of the active-site residue relative to the membrane surface, and (iii) fVa alters the orientation and/or the height of residue 195 above the membrane surface. 相似文献
94.
Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode
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95.
Rezaie Rezvan Rezaei Shahla Jafari Nasrin Forootanfar Hamid Khoshayand Mohammad Reza Faramarzi Mohammad Ali 《Extremophiles : life under extreme conditions》2017,21(6):993-1004
Extremophiles - Lignocellulose bioconversion is a harsh process requiring the use of surfactants and organic solvents. Consequently, the incorporation of laccases in this bioconversion requires the... 相似文献
96.
Background
Dab2, one of two mammalian orthologs of Drosophila Disabled, has been shown to be involved in cell positioning and formation of visceral endoderm during mouse embryogenesis, but its role in neuronal development is not yet fully understood. In this report, we have examined the localization of the Dab2 protein in the mouse embryonic central nervous system (CNS) at different developmental stages. 相似文献97.
98.
Angiogenic and Restorative Abilities of Human Mesenchymal Stem Cells Were Reduced Following Treatment With Serum From Diabetes Mellitus Type 2 Patients
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Jafar Rezaie Malek S. Mehranjani Reza Rahbarghazi Mohammad A. Shariatzadeh 《Journal of cellular biochemistry》2018,119(1):524-535
This experiment investigated the impact of serum from patients with type 2 diabetes mellitus on the angiogenic behavior of human mesenchymal stem cells in vitro. Changes in the level of Ang‐1, Ang‐2, cell migration, and trans‐differentiation into pericytes and endothelial lineage were monitored after 7 days. The interaction of mesenchymal stem cells with endothelial cells were evaluated using surface plasmon resonance technique. Paracrine restorative effect of diabetic stem cells was tested on pancreatic β cells. Compared to data from FBS and normal serum, diabetic serum reduced the stem cell survival and chemotaxis toward VEGF and SDF‐1α (P < 0.05). Diabetic condition were found to decline cell migration rate and the activity of MMP‐2 and ‐9 (P < 0.05). The down‐regulation of VEGFR‐2 and CXCR‐4 was observed with an increase in the level of miR‐1‐3p and miR‐15b‐5p at the same time. The paracrine angiogenic potential of diabetic stem cells was disturbed via the changes in the dynamic of Ang‐1, Ang‐2, and VEGF. Surface plasmon resonance analysis showed that diabetes could induce an aberrant increase in the interaction of stem cells with endothelial cells. After treatment with diabetic serum, the expression of VE‐cadherin and NG2 and ability for uptake of Dil‐Ac‐LDL were reduced (P < 0.01). Conditioned media prepared from diabetic stem cells were unable to decrease fatty acid accumulation in β‐cells (P < 0.05). The level of insulin secreted by β‐cells was not affected after exposure to supernatant from diabetic or non‐diabetic mesenchymal stem cells. Data suggest diabetes could decrease angiogenic and restorative effect of stem cells in vitro. J. Cell. Biochem. 119: 524–535, 2018. © 2017 Wiley Periodicals, Inc. 相似文献
99.
Ehsan?Rezaie Mozafar?MohammadiEmail author Amirhossein?Sakhteman Peyman?Bemani Sajjad?Ahrari 《Journal of molecular modeling》2018,24(11):313
Proteins are often monitored by combining a fluorescent polypeptide tag with the target protein. However, due to the high molecular weight and immunogenicity of such tags, they are not suitable choices for combining with fusion proteins such as immunotoxins. In this study, we designed a polypeptide sequence with a dual role (it acts as both a linker and a fluorescent probe) to use with fusion proteins. Two common fluorescent tag sequences based on tetracysteine were compared to a commonly used rigid linker as well as our proposed dual-purpose sequence. Computational investigations showed that the dual-purpose sequence was structurally stable and may be a good choice to use as both a linker and a fluorescence marker between two moieties in a fusion protein. 相似文献
100.
Masoud Nazarizadeh Hamid Reza Rezaie Jalil Imani Harisini Eric Pasquet 《Zoology in the Middle East.》2016,62(3):217-226
The Eurasian Nuthatch (Sitta europaea Linnaeus, 1758) is a resident bird in the Alborz and Zagros deciduous forests. We investigated the phylogenetic relationships and the taxonomic position of the Eurasian Nuthatch among other separated lineages of Eurasia with the help of blood samples collected from 19 individuals belonging to four populations in the Eastern and Western Alborz, as well as in the Northern and Southern Zagros forests. Genetic variation was then analysed using complete ND2 gene sequence (1041?bp) and phylogenetic analysis was done using Bayesian and maximum likelihood inference. Additionally, a median-joining algorithm was used to reveal the relationships among haplotypes. The results of the phylogenetic and haplo-type network analyses indicated that Eurasian Nuthatch haplotypes from the Alborz and Zagros Mountains form lineages distinct from the Asian, Caucasian and European haplotypes. Furthermore, an analysis of molecular variance (AMOVA) detected significant (P<0.001) genetic structure among the lineages. The Asian, European, Caucasian and Alborz lineages diverged from one another by an uncorrected genetic distance ranging from 0.029 to 0.039, while the Zagros lineage showed a slightly lower genetic divergence from the Caucasian lineage (0.006), but it did not share any haplotype with the Caucasian lineage. Thus, we suggest considering five Conservation Significant Units (CSU) for the Eurasian Nuthatches as the result of used dataset. 相似文献