共查询到20条相似文献,搜索用时 31 毫秒
1.
Background
Mutation in αA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of αA-crystallin and αB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of αA-crystallin on subunit exchange and interaction with αB-crystallin is unknown. In the present study, interaction of the mutants of αA-crystallin with αB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique.Methodology/Principal Findings
In vitro FRET technique was used to demonstrate the rates of subunit exchange of αB-wt with the following αA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with αB-wt decreased drastically as compared to αA-wt interacting with αB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of αA- mutants with αB-wt was also assessed by in situ FRET. YFP-tagged αA mutants were co-expressed with CFP-tagged αB-wt in HeLa cells and the spectral signals were captured with a confocal microscope before and after acceptor laser photobleaching. The interaction of R21W and R116C with αB-wt was decreased nearly 50% as compared to αA-wt while the rest of the mutants showed slightly decreased interaction. Thus, there is good agreement between the in vitro and in situ FRET data.Conclusions/Significance
Structural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with αB may contribute to development of congenital cataract. 相似文献2.
Shingo Harada Ryota Kajihara Risa Muramoto Promsuk Jutabha Naohiko Anzai Tetsuhiro Nemoto 《Bioorganic & medicinal chemistry letters》2018,28(10):1915-1918
p-Boronophenylalanine (l-BPA) is applied in clinical settings as a boron carrier for boron neutron capture therapy (BNCT) to cure malignant melanomas. Structural modification or derivatization of l-BPA, however, to improve its uptake efficiency into tumor cells has scarcely been investigated. We successfully synthesized (S)-2-amino-3-(4-boronophenyl)-2-methylpropanoic acid in enantioenriched form as a novel candidate molecule for BNCT. Key steps to enhance the efficiency of this synthesis were enantioselective alkylation of N-protected alanine tert-butyl ester with a Maruoka catalyst and Miyaura borylation reaction to install the boron functionality. 相似文献
3.
Luc Van Puyvelde Serge Dube Emmanuel Uwimana Celine Uwera Roger A. Dommisse Edgard L. Esmans Omer Van Schoor Arnold J. Vlietinck 《Phytochemistry》1979,18(7):1215-1218
The structures for umuravumbolide, 5,6-dihydro-6-(3-acetoxy-1-heptenyl)-2-pyrone, a new α-pyrone from Iboza riparia (Labiatae) and its corresponding deacylated product have been established. Deacetylboronolide was also isolated and identified by different spectroscopic techniques. 相似文献
4.
M. Thirunavukkarasu Fergus G. Priest 《Biochemical and biophysical research communications》1983,114(2):677-683
α-Glucosidase was membrane bound during exponential growth of but was released into the medium during stationary phase. It could be partially removed from exponential phase cells by washing with NaCl (0.5 M). α-Amylase was exclusively extracellular and could not be detected in cells. Polysomes were prepared from exponential phase cells and separated into membrane bound and soluble fractions. chain completion and immunoprecipitation showed that α-glucosidase and α-amylase were synthesized by membrane bound and not by soluble ribosomes. 相似文献
5.
Katsuko Yamashita Eiji Ichishima Masanori Arai Akira Kobata 《Biochemical and biophysical research communications》1980,96(3):1335-1342
The substrate specificity of an α-mannosidase purified from Aspergillus saitoi was studied in detail. This enzyme hydrolyzes yeast mannan partially but does not act on p-nitrophenyl α-mannopyranoside. Survey of the action of the enzyme on various oligosaccharides liberated from glycoproteins indicated that the enzyme hydrolyzes Manα1→2Man linkage but not Manα1→3Man and Manα1→6 Man linkages at all. All Manα1→2 residues in intact bovine pancreatic ribonuclease B were removed completely by incubation with the α-mannosidase. 相似文献
6.
7.
Growth of Escherichia coli on melibiose requires the induced synthesis of α-galactoside permease and α-galactosidase. Hydrolysis of the chromogenic substrate p-nitrophenyl-σ-galactoside by whole bacteria is dependent on intact oxidative metabolism. The α-galactosidase from E. coli was isolated for the first time as a soluble enzyme. In cell-free extracts p-nitrophenyl-α-galactoside hydrolisis was observed only at high protein concentrations and the activity decreased exponentially with the square of the dilution. The reason for this behaviour was shown to be that, unlike other known α-galactosidases, the enzyme of E. coli requires NAD. For optimal activity the enzyme also requires Mn2+, a high concentration of 2-mercaptoethanol, and a pH of 8.1. The approximate molecular weight of the active from of α-galactosidase as determined by sedimentation in a sucrose gradient is 200 000. Due to the instability of the enzyme, its purification has not been achieved. 相似文献
8.
This protocol describes a new approach for the preparation of α-allylated α,β-unsaturated carbonyl compound by chemoselective cross-coupling of propargyl alcohols with allyl carbonates using an unprecedented vanadium/palladium contemporaneous dual catalysis. This process involves 1,3-transposition of propargyl alcohols by an oxyvanadium catalyst to generate vanadium allenoates and the activation of allyl carbonates by a palladium catalyst to generate π-allylpalladium species. These two active intermediates trap each other more rapidly to afford the observed product, rather than being intercepted by the large excess of starting propargyl alcohol. One example for the preparation of this type of α-allylated α,β-unsaturated carbonyl compound is included in the text. It takes ~20 h to complete the protocol: 1.0 h to set up the reaction, 16 h for the reaction and 2.0 h for isolation and purification. This chemistry has been applied to obtain a wide range of α-allylated α,β-unsaturated ketones, esters and amides, which are highly valuable building blocks in organic synthesis. 相似文献
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10.
A trypsin inhibitor, isolated from whole-wheat grain (Triticum aestivum L.) by the method of biospecific chromatography on trypsin-Sepharose, was potent in inhibiting human salivary α-amylase. The bifunctional α-amylase/trypsin inhibitor was characterized by a narrow specificity for other α-amylases and proteinases. The high thermostability of the inhibitor was lost in the presence of SH group-reducing agents. The inhibitor-trypsin complex retained its activity against α-amylase. The inhibitor—α-amylase complex was active against trypsin. Studies of the enzyme kinetics demonstrated that the inhibition of α-amylase and trypsin was noncompetitive. Our results suggest the existence of two independent active sites responsible for the interaction with the enzymes. 相似文献
11.
Summary The influence of pH, temperature, substrate concentration and organic solvents (dimethylformamide, dimethylsulfoxide) on the -chymotrypsin stability in a water/organic solvent system was studied. The enzyme activity was measured as the dipeptide, AcPheLeuNH2 synthesis and the ester substrate hydrolysis. Enzyme stability was enhanced by lower pH and temperature values and higher substrate concentrations. Dimethylsulfoxide allowed an higher enzyme stability than dimethylformamide. -Chymotrypsin displayed an higher stability in the water medium when it was compared to the organic system. 相似文献
12.
Bentama A El Hadrami EM El Hallaoui A Elachqar A Lavergne JP Roumestant ML Viallefont P 《Amino acids》2003,24(4):423-426
alpha-Heterocyclic alpha-aminoesters were obtained in good yields by reaction of a glycine cation equivalent and different heterocyclic nucleophiles; diastereoselectivity using a carbohydrate (galactopyranose) as N-protecting group was modest. 相似文献
13.
Pingyang Liu Michael P. Torrens-Spence Haizhen Ding Bruce M. Christensen Jianyong Li 《Amino acids》2013,44(2):391-404
Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to β-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development. 相似文献
14.
N. Dalla Venezia R. Wilmotte L. Morlé A. Forissier N. Parquet M. Garbarz T. Rousset D. Dhermy N. Alloisio J. Delaunay 《Human genetics》1993,90(6):641-644
The 207 LeuPro mutation in spectrin has recently been identified as a cause of I/50-46a hereditary elliptocytosis (HE) or pyropoikilocytosis among Black people. We have found this mutation in a Moroccan family in both the heterozygous and homozygous states. The mutated -spectrin allele carried, in cis, the V/41 polymorphism, a common polymorphism altering the peptide maps and associated with a low-expression level. This is the first report of the cis combination of an HE mutation and the V/41 polymorphism. Presumably, such a combination accounts for the very low expression of the abnormal allele in the heterozygous state. 相似文献
15.
Yoshitake Sakae 《Molecular simulation》2013,39(4):302-310
We performed folding simulations of three proteins using four force fields, AMBER parm96, AMBER parm99, CHARMM 27 and OPLS-AA/L, in order to examine the features of these force fields. We studied three proteins, protein A (all α-helix), cold-shock protein (all β-strand) and protein G (α/β-structures), for the folding simulations. For the simulation, we used the simulated annealing molecular dynamics method, which was performed 50 times for each protein using the four force fields. The results showed that the secondary-structure-forming tendencies are largely different among the four force fields. AMBER parm96 favours β-bridge structures and extended β-strand structures, and AMBER parm99 favours α-helix structures and 310-helix structures. CHARMM 27 slightly favours α-helix structures, and there are also π-helix and β-bridge structures. OPLS-AA/L favours α-helix structures and 310-helix structures. 相似文献
16.
Aims: This study focused on the cloning, expression and characterization of recombinant α‐l ‐arabinosidases from Bifidobacterium longum H‐1. Methods and Results: α‐l ‐Arabinofuranosidase (AfuB‐H1) and bifunctional α‐l ‐arabinopyranosidase/β‐d ‐galactosidase (Apy‐H1) from B. longum H‐1 were identified by Southern blotting, and their recombinant enzymes were overexpressed in Escherichia coli BL21 (DE3). Recombinant AfuB‐H1 (rAfuB‐H1) was purified by single‐step Ni2+‐affinity column chromatography, whereas recombinant Apy‐H1 (rApy‐H1) was purified by serial Q‐HP and Ni2+‐affinity column chromatography. Enzymatic properties and substrate specificities of the two enzymes were assessed, and their kinetic constants were calculated. According to the results, rAfuB‐H1 hydrolysed p‐nitrophenyl‐α‐l ‐arabinofuranoside (pNP‐αL‐Af) and ginsenoside Rc, but did not hydrolyse p‐nitrophenyl‐α‐l ‐arabinopyranoside (pNP‐αL‐Ap). On the other hand, rApy‐H1 hydrolysed pNP‐αL‐Ap, p‐nitrophenyl‐β‐d ‐galactopyranoside (pNP‐βD‐Ga) and ginsenoside Rb2. Conclusions: Ginsenoside‐metabolizing bifidobacterial rAfuB‐H1 and rApy‐H1 were successfully cloned, expressed, and characterized. rAfuB‐H1 specifically recognized the α‐l ‐arabinofuranoside, whereas rApy‐H1 had dual functions, that is, it could hydrolyse both β‐d ‐galactopyranoside and α‐l ‐arabinopyranoside. Significance and Impact of the Study: These findings suggest that the biochemical properties and substrate specificities of these recombinant enzymes differ from those of previously identified α‐l ‐arabinosidases from Bifidobacterium breve K‐110 and Clostridium cellulovorans. 相似文献
17.
Iwona Stroynowski Anders Orn Robert S. Goodenow Minnie McMillan James Forman Peter R. Brayton Jeffrey Frelingers Leroy Hood 《Immunogenetics》1984,20(2):141-154
We have shown that cytotoxic T lymphocytes (CTL) raised in H-2
dmice use H-2Ld but not H-2Dd or H-2Kd antigens as restricting elements in lymphocytic choriomeningitis virus (LCMV) and vesicular stomatis virus (VSV) infections. To localize the regions of H-2Ld protein recognized by CTL, we constructed a recombinant H-2L
d/D
dgene encoding a hybrid antigen with 1 and 2 external domains of H-2Ld and 3, transmembrane and cytoplasmic domains of H-2Dd. The recombinant gene was transfected into mouse cells and the hybrid molecules were characterized serologically, biochemically and functionally. In all assays, H-2Ld/Dd molecules were recognized by LCMV- and VSV-specific H-2Ld-restricted CTL in a manner similar to that of wild-type H-2Ld antigens. Analogous results were obtained with alloreactive CTL. Hybrid antigens containing the 3 domain of H-2Ld fused to 1 and 2 domains of a Qa-2,3 region-encoded antigen were not used as restricting elements by LCMV-specific CTL. These results suggest that H-2Ld-restricted CTL directed against LCMV and VSV recognize determinants controlled by the 1 and/or 2 domains of the H-2Ld molecule.Abbreviations used in this paper CTL
cytotoxic T lymphocytes
- VSV
vesicular stomatitis virus
- LCMV
lymphocytic choriomeningitis virus
- tk
thymidine kinase
- HAT
hypoxanthine, aminopterine, thymidine
- HSV
herpes simplex virus
- FCS
fetal calf serum
- SAC
Staphylococcus aureus Cowan I strain
- TM
transmembrane
- CYT
cytoplasmic 相似文献
18.
Every cone snail produces a mixture of different conotoxins and secretes them to immobilize their prey and predators. α3/5 Conotoxins, isolated from fish-hunting cone snails, target muscle nicotinic acetylcholine receptors. The structure and function of α3/5 conotoxin from the piscivorous Conus achatinus have not been studied. We synthesized two pentadecamer peptides, Ac 1.1 a and Ac 1.1 b, with appropriate disulfide bonding, based on cDNA sequences of α3/5 conotoxins from C. achatinus. Ac 1.1 a and Ac 1.1 b differ by only one amino acid residue. They have similar potency on blocking recombinant mouse muscle acetylcholine receptor expressed in Xenopus laevis oocytes, with IC_(50) values of 36 nM and 26 nM, respectively. For Ac 1.1b, deletion of the first three N-terminal amino acids did not change its activity, indicating that the Nterminus is not involved in the interaction with its receptor. Furthermore, our experiments indicate that both toxins strongly prefer the α1-δ subunit interface instead of the α1-γ binding site on the mouse muscle nicotinic acetylcholine receptor. These peptides provide additional tools for the study of the structure and function of nicotinic receptor. 相似文献
19.
α-Bungarotoxin has been labeled with [3H]pyridoxamine phosphate, by reaction with pyridoxal phosphate followed by reduction with sodium boro[3H]hydride. Specific activities of up to 27 Ci/mmol have been obtained. Mono- and dilabeled toxins bind irreversibly to the acetylcholine receptor from Torpedo electroplax, despite a change in isoelectric point from 9.2 for native toxin to 6.2 for dilabeled toxin. The 3H-labeled α-bungarotoxin is usable for over a year. 相似文献
20.
T-lymphocyte-mediated immunosuppression has been described in several animal models and in man. In animal models, T-cell-mediated immunosuppression can hasten the development of cancers, permit the growth of tumors in immunocompetent hosts, and inhibit otherwise effective antitumor immunotherapy. Cyclophosphamide can abrogate the T-cell-mediated immunosuppression. However, inappropriately administered cyclophosphamide can adversely affect antitumor immunity. On the basis of data showing that interferon / (IFN/) and IFN selectively abrogate the T-cell-mediated dinitrofluorobenzene-specific suppressor function, we investigated the efficacy of purified murine IFN/ in manipulating tumorinduced T-cell-mediated immunosuppression in the wellcharacterized P815 mastocytoma model. In this model, generation of cytotoxicity in vitro and its inhibition by T cells correlates with antitumor immunity in vivo. We report that IFN/ selectively diminishes the generation of tumor-induced suppressor activity. 相似文献