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1.
AKR MuLV 70S RNA was separated on Poly(U)-Sepharose into poly(A) and non-poly(A) containing 34S subunits. The ratio of the two fractions was 2:1, respectively. Both fractions were hybridized to AKR MuLV [3H]cDNA, and the hybrids were assayed by nuclease S1 and cesium sulfate centrifugation. The poly(A) and non-poly(A) subunits hybridized to [3H]cDNA to the same extent (80%), with identical values; and the hybrids of both fractions had identical Tm values (81°C in 0.15 M NaCl). These results demonstrate that the poly(A) and non-poly(A) containing subunits of the AKR genome have identical or very similar base sequences in the heteropolymeric regions. 相似文献
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Iwao Kusaka 《生物化学与生物物理学报:生物膜》1974,345(1):62-73
In the presence of glucose in complex media, the following changes in the characters of the membranes of Bacillus subtilis and Bacillus cereus were observed. (1) The activity of succinate dehydrogenase and the amount of cytochromes of the membranes were greatly reduced. (2) The ratio of lipid to protein in the membranes was decreased and a membrane subfraction, which had a density of around 1.2 (B. subtilis) and 1.24 g/cm3 (B. cereus), was newly formed (B. subtilis) or increased (B. cereus). (3) The phospholipid and diglyceride contents in the membranes were reduced.Polyacrylamide gel electrophoresis of proteins of the two types (plus and minus glucose) of the membranes show that the patterns were very different between the two types of membranes, though the lost or newly formed membrane protein components were not observed.The cytochrome content was not increased when the cells were grown in glucose medium supplemented with haemin, therefore, glucose or its metabolite may not be involved in the inhibition of haem biosynthesis. 相似文献
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Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of Gi α-2, Gi α-3 and G-protein β-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of Gs α-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of Gs α-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 μM), GTP (100 μM), p[NH]ppG (100 μM), NaF (10 mM) and glucagon (10 μM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 μM) was lower by about 22%. Lower concentrations (EC50-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC50-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional Gi activity. The lower levels of expression of G-protein β-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals. 相似文献
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Judith Toneatto Guadalupe Lorenzatti Gerardo A. Argüello 《Journal of inorganic biochemistry》2010,104(7):697-2767
Due to the key role of DNA in cell life and pathological processes, the design of specific chemical nucleases, DNA probes and alkylating agents is an important research area for the development of new therapeutic agents and tools in Biochemistry. Hence, the interaction of small molecules with DNA has attracted in particular a great deal of attention.The aim of this study was to investigate the ability of [Cr(phen)2(dppz)]3+ to associate with DNA and to characterize it as photocleavage reagent for Photodynamic Therapy (PDT).Chromium(III) complex [Cr(phen)2(dppz)]3+, (dppz = dipyridophenazine, phen = 1,10-phenanthroline), where dppz is a planar bidentate ligand with an extended π system, has been found to bind strongly to double strand oligonucleotides (ds-oligo) and plasmid DNA with intrinsic DNA binding constants, Kb, of (3.9 ± 0.3) × 105 M−1 and (1.1 ± 0.1) × 105 M−1, respectively. The binding properties to DNA were investigated by UV-visible (UV-Vis) absorption spectroscopy and electrophoretic studies. UV-Vis absorption data provide clearly that the chromium(III) complex interacts with DNA intercalatively. Competitive binding experiments show that the enhancement in the emission intensity of ethidium bromide (EthBr) in the presence of DNA was quenched by [Cr(phen)2(dppz)]3+, indicating that the Cr(III) complex displaces EthBr from its binding site in plasmid DNA. Moreover, [Cr(phen)2(dppz)]3+, non-covalently bound to DNA, promotes the photocleavage of plasmid DNA under 457 nm irradiation. We also found that the irradiated Cr(III)-plasmid DNA association is able to impair the transforming capacity of bacteria. These results provide evidence confirming the responsible and essential role of the excited state of [Cr(phen)2(dppz)]3+ for damaging the DNA structure. The combination of DNA, [Cr(phen)2(dppz)]3+ and light, is necessary to induce damage. In addition, assays of the photosensitization of transformed bacterial suspensions suggest that Escherichia coli may be photoinactivated by irradiation in the presence of [Cr(phen)2(dppz)]3+. In sum, our results allow us to postulate the [Cr(phen)2(dppz)]3+ complex as a very attractive candidate for DNA photocleavage with potential applications in Photodynamic Therapy (PDT). 相似文献
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Phosphorylation of ADP and nucleotide exchange by membrane-bound coupling factor CF1 are very fast reactions in the light, so that a direct comparison of both reactions is difficult. By adding substrate ADP and phosphate to illuminated thylakoids together with the uncoupler FCCP, the phosphorylation time is limited and the amount of ATP formed can be reduced to less than 1 ATP per enzyme. Low concentrations of medium nucleotides during illumination increase the amount of ATP formed during uncoupling presumably by binding to the tight nucleotide binding site (further designated as ‘site A’) with an affinity of 1 to 7 μM for ADP and ATP. ATP formation itself shows half-saturation at about 30 μM. Loosely bound nucleotides are exchanged upon addition of nucleotides with uncoupler (Schumann, J. (1984) Biochim. Biophys. Acta 766, 334–342). Release depends binding of nucleotides to a second site. The affinity of this site for ADP (in the presence of phosphate) is about 30 μM. It is assumed that phosphorylation and induction of exchange both occur on the same site (site B). During ATP hydrolysis, an ATP molecule is bound to site A, while on another site, ATP is hydrolyzed rapidly. The affinity of ADP for the catalytic site (70 μM) is in the same range as the observed Michaelis constant of ADP during phosphorylation; it is assumed that site B is involved in ATP hydrolysis. Site A exhibits some catalytic activity; it might be that site A is involved in ATP formation in a dual-site mechanism. For ATP hydrolysis, however, direct determination of exchange rates showed that the exchange rate of ATP bound to site A is about 30-times lower than ATP hydrolysis under the same conditions. 相似文献
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Carlos Velez-Pardo Marlene Jimenez del Rio Guy Ebinger Georges Vauquelin 《Neurochemistry international》1995,26(6):615-622
The authors previously reported that Fe2+ is capable of increasing the binding of dopamine and of serotonin to “serotonin binding proteins” which are present in soluble extracts from calf brain. In this study, it is shown that Mn2+ and Cu2+ are also capable of increasing the binding, but for dopamine only. As for Fe2+, Mn2+ and Cu2+ are likely to promote the binding by virtue of their ability to enhance the oxidation of dopamine into dopamine-O-quinone, a derivative which is known to undergo covalent association with sulfhydryl groups of proteins. Data such as the irreversible nature of the majority of the binding, the inhibitory action of reducing agents (sodium ascorbate) and of reagents which contain, or modify sulfhydryl groups (reduced glutathione) are compatible with such a mechanism. The three metal ions are also capable of inactivating part of the binding sites on SBP directly; this effect is more pronounced for Cu2+ than for Fe2+ and it is only weak for Mn2+. The Fe2+-mediated binding of dopamine is inhibited by the superoxide dismutase enzyme, and it was therefore suggested that Fe2+ enhances the oxidation of dopamine by virtue of its ability to produce superoxide radicals out of dissolved molecular oxygen. Such a mechanism does not appear to take place in the case of Mn2+ and Cu2+. Instead, it is likely that Cu2+ and dopamine form a complex which is highly susceptible towards oxidation by dissolved molecular oxygen. Mn2+, on the other hand, can easily be oxidized into Mn3+, which is capable to oxidize dopamine by itself. Chronic manganese intoxication (from exposure to manganese) and Wilson's disease (related to inadequate elimination of copper) go along with neurological symptoms which are very similar to those encountered in Parkinson's disease. Our data indicate that manganese and copper ions accelerate the oxidation of catecholamines to produce toxic quinones. These quinones could, at least in part, account for the degeneration of dopamininergic neurons in such pathologies. 相似文献
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Damien Keogh Roisin Thompson Ruth Larragy Kenneth McMahon Michael O'Connell Brendan O'Connor Paul Clarke 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Prokaryotic lectins offer significant advantages over eukaryotic lectins for the development of enhanced glycoselective tools. Amenability to recombinant expression in Escherichia coli simplifies their production and presents opportunities for further genetic manipulation to create novel recombinant prokaryotic lectins (RPLs) with altered or enhanced carbohydrate binding properties. This study explored the potential of the α-galactophilic PA-IL lectin from Pseudomonas aeruginosa for use as a scaffold structure for the generation of novel RPLs.Method
Specific amino acid residues in the carbohydrate binding site of a recombinant PA-IL protein were randomly substituted by site-directed mutagenesis. The resulting expression clones were then functionally screened to identify clones expressing rPA-IL proteins with altered carbohydrate binding properties.Results
This study generated RPLs exhibiting diverse carbohydrate binding activities including specificity and high affinity for β-linked galactose and N-acetyl-lactosamine (LacNAc) displayed by N-linked glycans on glycoprotein targets. Key amino acid substitutions were identified and linked with specific carbohydrate binding activities. Ultimately, the utility of these novel RPLs for glycoprotein analysis and for selective fractionation and isolation of glycoproteins and their glycoforms was demonstrated.Conclusions
The carbohydrate binding properties of the PA-IL protein can be significantly altered using site-directed mutagenesis strategies to generate novel RPLs with diverse carbohydrate binding properties.General significance
The novel RPLs reported would find a broad range of applications in glycobiology, diagnostics and in the analysis of biotherapeutics. The ability to readily produce these RPLs in gram quantities could enable them to find larger scale applications for glycoprotein or biotherapeutic purification. 相似文献11.
Janvilisri T Likitponrak W Chunchob S Grams R Vichasri-Grams S 《Molecular and cellular biochemistry》2007,305(1-2):95-102
Fatty acid binding proteins (FABPs) are capable of binding hydrophobic ligands with high affinity; thereby facilitating the
cellular uptake and intracellular trafficking of fatty acids. In this study, functional characteristics of a cytoplasmic FABP
from the giant liver fluke Fasciola gigantica (FgFABP) were determined. Binding of a fluorescent fatty acid analogue 11-[[5-dimethy aminonaphtalene-1-sulphonyl] amino]
undecanoic acid (DAUDA) to FgFABP resulted in changes in the emission spectrum. The optimal excitation wavelength and maximum
emission of fluorescence for binding activities with DAUDA were 350 nm and 550 nm, respectively. The binding activity for
DAUDA was determined from titration experiments and revealed a Kd value of 2.95 ± 0.54 μM. Furthermore, we found that cross-linking profile of FgFABP with dithiobis-(succinimidylpropionate)
(DSP) in the presence of DAUDA resulted in increased formation of higher-ordered oligomers compared to that in the absence
of DAUDA. We also replaced five highly conserved positively charged residues (K9, K58, K91, R107 and K131) with alanine and
studied their oligomerization and binding properties of the modified FgFABPs. The obtained data demonstrate that these residues
do not appear to be involved in oligomerization. However, the K58A and R107A substitutions exhibited a reduction in binding
affinities. K91A and R107A revealed an increase in maximal specific binding. 相似文献
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The synergistic effect of steam explosion pretreatment and sodium hydroxide post-treatment of Lespedeza stalks (Lespedeza crytobotrya) has been investigated in this study. In this case, Lespedeza stalks were firstly exploded at a fixed steam pressure (22.5 kg/m2) for 2–10 min. Then the steam-exploded Lespedeza stalks was extracted with 1 M NaOH at 50 °C for 3 h with a shrub to water ratio of 1:20 (g/ml), which yielded 57.3%, 53.1%, 55.4%, 52.8%, 53.2%, and 56.4% (% dry weight) cellulose rich fractions, comparing to 68.0% from non-steam-exploded material. The content of glucose in cellulose rich residues increased with increment of the steaming time and reached to 94.10% at the most severity. The similar increasing trend occurred during the dissolution of hemicelluloses. It is evident that at shorter steam explosion time, autohydrolysis mainly occurred on the hemicelluloses and the amorphous area of cellulose. The crystalline region of cellulose was depolymerized under a prolonged incubation time. The characteristics of the cellulose rich fractions in terms of FT-IR and CP/MAS 13C NMR spectroscopy and thermal analysis were discussed, and the surface structure was also investigated by SEM. 相似文献
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Xiaoyun Wu Yuanyuan Wang Shanhe Wan 《Journal of biomolecular structure & dynamics》2013,31(12):3106-3113
The c-ros oncogene 1 (ROS1) has proven to be an important cancer target for the treatment of various human cancers. The anaplastic lymphoma kinase inhibitor crizotinib has been granted approval for the treatment of patients with ROS1 positive metastatic non-small-cell lung cancer by the Food and Drug Administration on 2016. However, serious resistance due to the secondary mutation of glycine 2032 to arginine (G2032R) was developed in clinical studies. Loratinib (PF-06463922), a macrocyclic analog of crizotinib, showed significantly improved inhibitory activity against wild–type (WT) ROS1 and ROS1G2032R mutant. To provide insights into the inhibition mechanism, molecular dynamics simulations and free energy calculations were carried out for the complexes of loratinib with WT and G2032R mutated ROS1. The apo-ROS1WT and apo-ROS1G2032R systems showed similar RMSF distributions, while ROS1G2032R-loratinib showed significantly higher than that of WT ROS1-loratinib, which revealed that the binding of loratinib to ROS1G2032R significantly interfered the ?uctuation of protein. Calculations of binding free energies indicate that G2032R mutation significantly reduces the binding affinity of loratinib for ROS1, which arose mostly from the increase of conformation entropy and the decrease of solvation energy. Furthermore, detailed per-residue binding free energies highlighted the increased and decreased contributions of some residues in the G2032R mutated systems. The present study revealed the detailed inhibitory mechanism of loratinib as potent WT and G2032R mutated ROS1 inhibitor, which was expected to provide a basis for rational drug design. 相似文献
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Carl Tannenbaum Gerhard Toggenburger Markus Kessler Aser Rothstein Giorgio Semenza 《Journal of cellular biochemistry》1977,6(4):519-533
In the presence of an NaSCN gradient phlorizin binds with a high affinity (Kd ? 4.7 μM) to vesicles derived from brush border membranes of intestinal cells of rabbits. The value for Kd corresponds closely to that of Ki determined from phlorizin inhibition of sugar transport. The apparent affinity for phlorizin is decreased if NaCl is substituted for NaSCN and decreased substantially if the gradient of NaSCN is allowed to dissipate prior to the phlorizin binding. The number of high affinity binding sites is about 11 pmol/mg protein. Additional binding to low affinity sites can amount to as much as 600 pmol/mg protein after prolonged exposure to phlorizin (5 min). The high affinity sites are related to glucose transport based on the similarity of the Kd and Ki values under a variety of conditions and on the inhibition of the binding by D-glucose but not by D-fructose. The transport system and the high affinity phlorizin binding sites can be enriched by a factor of 2–3 by treatment of vesicles with papain, which does not affect the transport system, but considerably hydrolyzes nonrelevant protein. 相似文献
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Celia Bonaventura Robert Henkens Walleska De Jesus-Bonilla Juan Lopez-Garriga Yiping Jia Abdu I. Alayash Claire J. Parker Siburt Alvin L. Crumbliss 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(10):1988-1995
The clam Lucina pectinalis supports its symbiotic bacteria by H2S transport in the open and accessible heme pocket of Lucina Hb I and by O2 transport in the narrow and crowded heme pocket of Lucina Hb II. Remarkably, air-equilibrated samples of Lucina Hb I were found to be more rapidly oxidized by nitrite than any previously studied Hb, while those of Lucina Hb II showed an unprecedented resistance to oxidation induced by nitrite. Nitrite-induced oxidation of Lucina Hb II was enabled only when O2 was removed from its active site. Structural analysis revealed that O2 “clams up” the active site by hydrogen bond formation to B10Tyr and other distal-side residues. Quaternary effects further restrict nitrite entry into the active site and stabilize the hydrogen-bonding network in oxygenated Lucina Hb II dimers. The dramatic differences in nitrite reactivities of the Lucina Hbs are not related to their O2 affinities or anaerobic redox potentials, which were found to be similar, but are instead a result of differences in accessibility of nitrite to their active sites; i.e. these differences are due to a kinetic rather than thermodynamic effect. Comparative studies revealed heme accessibility to be a factor in human Hb oxidation by nitrite as well, as evidenced by variations of rates of nitrite-induced oxidation that do not correlate with R and T state differences and inhibition of oxidation rate in the presence of O2. These results provide a dramatic illustration of how evolution of active sites with varied heme accessibility can moderate the rates of inner-sphere oxidative reactions of Hb and other heme proteins. 相似文献
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Fluorescence induction curves in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited Photosystem (PS) II particles isolated from the blue-green alga Phormidium laminosum have been analysed as a function of redox potential. Redox titration of the initial fluorescence indicated a single component with Em,7.5 = +30 mV (n = 1) (Bowes, J., Horton, P. and Bendall, D.S. (1981) FEBS Lett. 135, 261–264). Despite this simplified electron acceptor system and the small number of chlorophylls per reaction centre, a sigmoidal induction curve was nevertheless seen. Sigmoidicity decreased as Q was reduced potentiometrically prior to induction such that the induction was exponential when the ratio . These particles also showed a slow (β) phase of induction which titrated with an Em value slightly more positive than that of the major quencher. It is concluded that the sigmoidal shape of the fluorescence induction curve observed in Phormidium PS II particles is not a consequence of a requirement for two photons to close the PS II reaction centre, but is generated as a result of energy transfer between photosynthetic units comprising one reaction centre per approx. 50 chlorophylls. Also, the existence of PS II heterogeneity (PS IIα, PS IIβ centres) does not require a structurally differentiated chloroplast, but may only indicate the extent of aggregation of PS II centres. 相似文献
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The interaction of (−)-reboxetine, a non-tricyclic norepinephrine selective reuptake inhibitor, with muscle-type nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that (−)-reboxetine: (a) inhibits (±)-epibatidine-induced Ca2+ influx in human (h) muscle embryonic (hα1β1γδ) and adult (hα1β1εδ) AChRs in a non-competitive manner and with potencies IC50 = 3.86 ± 0.49 and 1.92 ± 0.48 μM, respectively, (b) binds to the [3H]TCP site with ∼13-fold higher affinity when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [3H]cytisine binding to the resting but activatableTorpedo AChR but not to the desensitized AChR, suggesting desensitizing properties, (d) overlaps the PCP luminal site located between rings 6′ and 13′ in the Torpedo but not human muscle AChRs. In silico mutation results indicate that ring 9′ is the minimum structural component for (−)-reboxetine binding, and (e) interacts to non-luminal sites located within the transmembrane segments from the Torpedo AChR γ subunit, and at the α1/ε transmembrane interface from the adult muscle AChR. In conclusion, (−)-reboxetine non-competitively inhibits muscle AChRs by binding to the TCP luminal site and by inducing receptor desensitization (maybe by interacting with non-luminal sites), a mechanism that is shared by tricyclic antidepressants. 相似文献
18.
The proteins encoded by psaA and psaB form a heterodimer, an essential compound of photosystem; while the protein encoded by psbC binds with chlorophyll a in photosystem II, serving as antennae in photosystem. Here we report that a heterocyclic brominated flame retardant, tris(2,3-dibromopropyl) isocyanurate (TBC), inhibited the expression of psaA and psbC, then leads to the decrease of Nannochloropsis sp.'s growth biomass. TBC exposure inhibited the expression of psaA and psbC at 10, 100 ng/mL slightly and 1000 ng/mL significantly. In addition, TBC was found to slow down the growth of Nannochloropsis sp. at concentrations ranging from 10 to 1000 ng/mL. These results indicated that TBC influenced both photosynthesis and growth performance of Nannochloropsis sp. 相似文献
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Gilberto Vargas-Alarcon Maite Vallejo Carlos Posadas-Romero Juan G. Juarez-Rojas Marco Antonio Martinez-Rios Marco Antonio Peña-Duque Silvia Carrillo-Sanchez Oscar Perez-Mendez José Manuel Fragoso 《Gene》2014
Endothelial dysfunction plays an essential role in the development and progression of atherosclerotic lesions. Endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) are considered important molecules in the endothelial dysfunction process. The aim of the present study was to evaluate the role of eNOS and ET-1 (EDN1) gene polymorphisms as susceptibility markers for acute coronary syndrome (ACS). Six polymorphisms (rs1799983, rs2070744, rs1800783, rs3087459, rs1800541, and rs5369) of eNOS and EDN1 genes were analyzed by 5′ exonuclease TaqMan genotyping assays in a group of 452 patients with ACS and 283 healthy controls. The results showed increased frequencies of the A allele of the END1-914 C>A (rs3087459) polymorphism in ACS patients when compared to controls (OR = 1.56, Pc = 0.01). Under an additive model, the “AA” genotype was associated with an increased risk of developing ACS, adjusted for gender, hypertension, dyslipidemia, alcohol consumption, smoking, and diabetes (OR = 1.56, p = 0.045). Linkage disequilibrium analysis showed one EDN1 haplotype (AT) with increased frequency in ACS patients when compared to healthy controls (OR = 1.65, Pc = 0.0015). The “AT” haplotype was associated with the risk of developing ACS after adjusting for cardiovascular risk factors using multiple logistic analysis. In this case, the adjusted OR was 1.73 for the AT haplotype (Pc = 0.0018). In summary, resulting data suggest that the END1-914 C>A gene polymorphism could be involved in the risk of developing ACS in Mexican individuals. 相似文献
20.
E. Carboni M. Memo G.L. Tanda M.O. Carruba P.F. Spano 《Neurochemistry international》1985,7(2):279-284
Sulpiride is an antipsychotic drug endowed with the properties of a dopamine antagonist. The failure of sulpiride to inhibit neostriatal dopamine stimulated adenylate cyclase activity indicated that this drug is a selective D2 receptor antagonist. In this study we used a novel synthesized 2H(—)sulpiride with very high specific activity (72 Ci/mol) and characterized the temperature sensitivity of the binding sites labeled by this compound. Kinetic analysis of 3H(—)sulpiride binding in rat striatum showed unstable behavior when incubation was performed at 37 or 30°C. However when experiments were carried out at 15 or 10°C, binding reached a stable steady-state within 10 min. Scatchard analysis of binding isotherms obtained at 10°C showed a 5-fold increase in the maximum number of binding sites and a decrease in Kd values to one-third those obtained at 37°C. Pharmacological characterization of the binding sites labeled by 3H(—)sulpiride at 10°C showed a greater affinity for antagonists but not for agonists than 37°C. Under both experimental condition, 3H(—)sulpiride binding sites were Na+ and GTP-sensitive. The temperature sensitive binding phenomenon appeared to be area specific. 3H(—)sulpiride binding sites in tissues other than from striatum were influenced less or not at all by changes in incubation temperature. 相似文献