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991.
Sengupta R Sahoo R Ray SS Dutta T Dasgupta A Ghosh S 《Molecular and cellular biochemistry》2006,284(1-2):117-126
The oxygenase domain of the inducible nitric oxide synthase, Δ65 iNOSox is a dimer that binds heme, L-Arginine (L-Arg), and tetrahydrobiopterin (H4B) and is the site for NO synthesis. The role of H4B in iNOS structure-function is complex and its exact structural role is presently unknown. The present paper provides a simple
mechanistic account of interaction of the cofactor tetrahydrobiopterin (H4B) with the bacterially expressed Δ65 iNOSox protein. Transverse urea gradient gel electrophoresis studies indicated the presence
of different conformers in the cofactor-incubated and cofactor-free Δ65 iNOSox protein. Dynamic Light Scattering (DLS) studies
of cofactor-incubated and cofactor-free Δ65 iNOSox protein also showed two distinct populations of two different diameter
ranges. Cofactor tetrahydrobiopterin (H4B) shifted one population, with higher diameter, to the lower diameter ranges indicating conformational changes. The additional
role played by the cofactor is to elevate the heme retaining capacity even in presence of denaturing stress. Together, these
findings confirm that the H4B is essential in modulating the iNOS heme environment and the protein environment in the dimeric iNOS oxygenase domain. (Mol
Cell Boichem xxx: 1–10, 2005)
Supported by Calcutta University Research Grants. 相似文献
992.
Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations. 相似文献
993.
Li SL Yamamoto T Yoshimoto T Uchihi R Mizutani M Kurimoto Y Tokunaga K Jin F Katsumata Y Saitou N 《Human genetics》2006,118(6):695-707
We have analyzed 105 autosomal polymorphic short tandem repeat (STR) loci for nine East and South-eastern Asian populations
(two Japanese, five Han Chinese, Thai, and Burmese populations) and a Caucasian population using a multiplex PCR typing system.
All the STR loci are genomewide tetranucleotide repeat markers of which the total number of observed alleles and the observed
heterozygosity were 756 and 0.743, respectively, for Japanese populations. Phylogenetic analysis for these allele frequency
data suggested that the Japanese populations are more closely related with southern Chinese populations than central and/or
northern ones. STRUCTURE program analysis revealed the almost clearly divided and accountable population structure at K=2–6, that the two Japanese populations always formed one group separated from the other populations and never belong to different
groups at K≥3. Furthermore, our new allele frequency data for 91 loci were analyzed with those for 52 worldwide populations published
by previous studies. Phylogenetic and multidimensional scaling (MDS) analyses indicated that Asian populations with large
population size (six Han Chinese, three Japanese, two Southeast Asia) formed one distinct cluster and are closer to each other
than other ethnic minorities in east and Southeast Asia. This pattern may be the caviar of comparing populations with greatly
differing population sizes when STR loci were analyzed. 相似文献
994.
Wall-associated kinase 1--WAK1 is a transmembrane protein containing a cytoplasmic Ser/Thr kinase domain and an extracellular domain in contact with the pectin fraction of the plant cell wall in Arabidopsis thaliana (L.) HEYNH. In a previous paper [Decreux, A., Messiaen, J., 2005. Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation. Plant Cell Physiol. 46, 268-278], we showed that a recombinant peptide expressed in yeast corresponding to amino acids 67-254 of the extracellular domain of WAK1 specifically interacts with commercial non-methylesterified homogalacturonic acid, purified homogalacturonans from Arabidopsis and oligogalacturonides in a calcium-induced conformation. In this report, we used a receptor binding domain sequence-based prediction method to identify four putative binding sites in the extracellular domain of WAK1, in which cationic amino acids were selected for substitution by site-directed mutagenesis. Interaction studies between mutated forms of WAK1 and homogalacturonans allowed us to identify and confirm at least five specific amino acids involved in the interaction with homogalacturonan dimers and multimers. The presence of this homogalacturonan-binding domain within the extracellular domain of WAK1 is discussed in terms of cell wall architecture and signal transduction. 相似文献
995.
996.
Interaction selection by biopanning from a fragmented yeast proteome displayed on filamentous phage particles was successful in identifying proline-rich fragments of Boi1p and Boi2p. These proteins bind to the second ``src homology region 3' (SH3) domain of Bem1p, a protein of Saccharomyces cerevisiae involved in bud formation. Target Bem1p was a doubly-tagged recombinant, Bem1[Asn142-Ile551], which strongly interacts in ELISA with a C-terminal 75 amino acids polypeptide from Cdc24p exposed on phage. The whole yeast genomic display library contained ~7.7 × 107 independent clones of sheared S. cerevisiae genomic DNA fused to a truncated M13 gene III. This study corroborates the value of fragmented-proteome display to identify strong and direct interacting protein modules. 相似文献
997.
The glycoprotein B (gB) of human cytomegalovirus represents a dominant antigen for the humoral immune response. The immunodominant
region on gB is the antigenic domain 1 (AD-1), a complex structure that requires a minimal continuous sequence of more than
75 amino acids for antibody binding. In this study, this domain was expressed in Escherichia coli as a fusion protein with β-galactosidase but yielded insoluble protein aggregates as inclusion bodies. To recover the fusion
protein, inclusion bodies were solubilized by two extractions with urea 8 m and the fusion protein then isolated using gel filtration chromatography. After confirmation of fusion protein antigenicity
by Western blotting, the purified product was used as the capturing antigen in an enzyme-linked immunosorbent assay (ELISA)
to determine the presence of viral antibodies in serum samples of pregnant women. A cut-off point of approximately 0.2 absorbance
units could discriminate the results of seropositive from seronegative pregnant women. The data indicates the potential usefulness
of the fusion protein for the development of immunoassay for detection of the HCMV antibodies.
Received 22 September 2005; Revisions requested 10 October 2005; Revisions received 26 October 2005; Accepted 31 October 2005 相似文献
998.
The p75(NTR) tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells 总被引:7,自引:0,他引:7
Khwaja F Tabassum A Allen J Djakiew D 《Biochemical and biophysical research communications》2006,341(4):1184-1192
The p75 neurotrophin receptor (p75(NTR)) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75(NTR) retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (DeltaDD) dominant-negative antagonist of p75(NTR) showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75(NTR)-dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75(NTR) expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75(NTR) rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75(NTR) was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75(NTR)-dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75(NTR) expressing prostate cancer cells. 相似文献
999.
Histone deacetylase 6 (HDAC6) is the only known HDAC with two potentially functional catalytic domains, yet the role towards substrate played by these two domains remains ambiguous. Most studies report HDAC6 activities measured using either immune complexes or in vitro translated products. Here, we characterize the activity of highly purified recombinant HDAC6, mutants with active site histidine mutations in each domain (H216A and H611A), and individual catalytic domains. The deacetylase activities of these proteins, as well as their kinetic parameters, were measured using histone, alpha-tubulin, and fluorogenic acetylated lysine as substrates. Mutant H216A only slightly lowers the catalytic rate. However, mutant H611A decreases the catalytic rate more than 5000-fold. The first domain expressed alone is not catalytically active. In contrast, the second domain shows only a modest decrease in substrate binding and product formation rate. Our results indicate that the in vitro deacetylase activity of HDAC6 resides in the C-terminal second catalytic domain. 相似文献
1000.
Distinct Rab27A binding affinities of Slp2-a and Slac2-a/melanophilin: Hierarchy of Rab27A effectors
Fukuda M 《Biochemical and biophysical research communications》2006,343(2):666-674
The small GTPase Rab27A has recently been shown to regulate melanosome transport in mammalian skin melanocytes through sequentially interacting with two Rab27A effectors, Slac2-a/melanophilin and Slp2-a. Although Slac2-a and Slp2-a contain a similar N-terminal Rab27A-binding domain (named SHD, Slp homology domain), nothing is known about the functional differences between the Slac2-a SHD and Slp2-a SHD. In this study, the Rab27A-binding affinity of ten putative Rab27A effector proteins has been investigated. It has been found that they could be classified into a low-affinity group (e.g., Slac2-a) and a high-affinity group (e.g., Slp2-a and Slp4-a) based on their Rab27A-binding affinity. Kinetic analysis of the GTP-Rab27A-binding to the SHD of Slp2-a, Slp4-a, and Slac2-a by surface plasmon resonance further indicated that the kinetic parameters of Rab27A for the Slp2-a SHD, Slp4-a SHD, and Slac2-a SHD consisted of a fast association rate constant (3.35 x 10(4), 2.06 x 10(4), and 2.11 x 10(4) M(-1) s(-1), respectively) and a slow dissociation rate constant (4.48 x 10(-4), 3.96 x 10(-4), and 2.37 x 10(-3) s(-1) respectively), and their equilibrium dissociation constants were determined to be 13.4, 19.2, and 112 nM, respectively. Our data suggest that distinct Rab27A binding activities of Slac2-a and Slp2-a ensure the order (or hierarchy) of Rab27A effectors that sequentially function in melanosome transport in melanocytes. 相似文献