951 resultados para Binding sites (Biochemistry)


Relevância:

100.00% 100.00%

Publicador:

Resumo:

Restriction enzyme inhibition and lambda exonuclease studies indicate that carcinogen N-acetoxy-N-acetyl-2 aminofluorene (AAAF) binds to sequences on ɸX174 RF and SV40 plasmids DNA that are similar to the eight preferred binding sites previously located on pBR 322. Both DNAs were digested with enzyme Hinf I and resultant fragments 32P end-labeled. Labeled fragments were reacted with the carcinogen to give one to sixteen bound moieties per DNA. Fragments were isolated and restriccion enzyme and lambda exonuclease inhibition assays were performed. Inhibition detected occurred at selected sites and was not specific for a certain enzyme or certain size of recognition sequence. Results of these assays allow mapping of the location of high affinity binding sites of the carcinogen on both DNAs. All sites have common sequence elements: the presence of either the sequence T(G/C)TT(G/C) or the sequence T(G/C) CTT(G/C).

Relevância:

100.00% 100.00%

Publicador:

Resumo:

The ontogeny of muscarinic receptors was studied in human fetal striatum, brainstem, and cerebellum to investigate general principles of synaptogenesis as well as the physiological balance between various chemical synapses during development in a given region of the brain. [3H]Quinuclidinyl benzilate ([-'H]QNB) binding was assayed in total particulate fraction (TPF) from various parts of brain. In the corpus striatum, QNB binding sites are present at 16 weeks of gestation (average concentration 180 fmol/mg protein of TPF), slowly increase up to 24 weeks (average concentration 217 fmol/mg protein), and rapidly increase during the third trimester to 480 fmol/mg protein of TPF. In contrast, dopaminergic receptors exist as two subpopulations. one with low affinity and the other with high affinity up to the 24th week of gestation; all of them acquire the highaffinity characteristic during the third trimester. In brainstem, the muscarinic receptors show maximum concentration by 16 weeks of age (360 fmolimg protein of TPF). Subsequently the muscarinic receptor concentration shows a gradual decline in the brainstem. In cerebellum, except for a slight increase at 24 weeks (average concentration 90 fmol/mg protein of TPF), the receptor concentration remained nearly constant at about 60-70 fmolimg protein of TPF throughout fetal life. This study demonstrates that the ontogeny of muscarinic receptors varies among the different regions, and the patterns observed suggest that receptor formation occurs principally in the third trimester. Also noteworthy is the finding that the QNB binding sites decreased in all regions of the human brain during adult life. Key Words: Cholinergic muscarinic receptors-Quinuclidinyl benzilate-Corpus striaturn-Brainstem-Cerebellum. Ravikumar B. V. and Sastry P. S. Cholinergic muscarinic receptors in human fetal brain: Ontogeny of [3H]quinuclidinyl benzilate binding sites in corpus striatum, brainstem, and cerebellum. J. Neurochem. 45, 1948- 1950 (1985).

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Eighteen corpora striata from normal human foetal brains ranging in gestational age from 16 to 40 weeks and five from post natal brains ranging from 23 days to 42 years were analysed for the ontogeny of dopamine receptors using [3H]spiperone as the ligand and 10 mM dopamine hydrochloride was used in blanks. Spiperone binding sites were characterized in a 40-week-old foetal brain to be dopamine receptors by the following criteria: (1) It was localized in a crude mitochondrial pellet that included synaptosomes; (2) binding was saturable at 0.8 nM concentration; (3) dopaminergic antagonists spiperone, haloperidol, pimozide, trifluperazine and chlorpromazine competed for the binding with IC50 values in the range of 0.3–14 nM while agonists—apomorphine and dopamine gave IC50 values of 2.5 and 10 μM, respectively suggesting a D2 type receptor.Epinephrine and norepinephrine inhibited the binding much less efficiently while mianserin at 10 μM and serotonin at 1 mM concentration did not inhibit the binding. Bimolecular association and dissociation rate constants for the reversible binding were 5.7 × 108 M−1 min−1 and 5.0 × 10−2 min−1, respectively. Equilibrium dissociation constant was 87 pM and the KD obtained by saturation binding was 73 pM.During the foetal age 16 to 40 weeks, the receptor concentration remained in the range of 38–60 fmol/mg protein or 570–1080 fmol/g striatum but it increased two-fold postnatally reaching a maximum at 5 years Significantly, at lower foetal ages (16–24 weeks) the [3H]spiperone binding sites exhibited a heterogeneity with a high (KD, 13–85 pM) and a low (KD, 1.2–4.6 nM) affinity component, the former accounting for 13–24% of the total binding sites. This heterogeneity persisted even when sulpiride was used as a displacer. The number of high affinity sites increased from 16 weeks to 24 weeks and after 28 weeks of gestation, all the binding sites showed only a single high affinity.GTP decreased the agonist affinity as observed by dopamine competition of [3H]spiperone binding in 20-week-old foetal striata and at all subsequent ages. GTP increased IC50 values of dopamine 2 to 4.5 fold and Hill coefficients were also increased becoming closer to one suggesting that the dopamine receptor was susceptible to regulation from foetal life onwards.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Expression of genes involved in methanol metabolism of Pichia pastoris is regulated by Mxr1p, a zinc finger transcription factor. In this study, we studied the target gene specificity of Mxr1p by examining its ability to bind to promoters of genes encoding dihydroxyacetone synthase (DHAS) and peroxin 8 (PEX8), since methanol-inducible expression of these genes is abrogated in mxr1-null mutant strains of P. pastoris. Different regions of DHAS and PEX8 promoter were isolated from P. pastoris genomic DNA and their ability to bind to a recombinant Mxr1p protein containing the N-terminal 150 amino acids, including the zinc finger DNA-binding domain, was examined. These studies reveal that Mxr1p specifically binds to promoter regions containing multiple 5'-CYCC-3' sequences, although all DNA sequences containing the 5'-CYCC-3' motif do not qualify as Mxr1p-binding sites. Key DNA-binding determinants are present outside 5'-CYCC-3' motif and Mxr1p preferably binds to DNA sequences containing 5'-CYCCNY-3' than those containing 5'-CYCCNR-3' sequences. This study provides new insights into the molecular determinants of target gene specificity of Mxr1p, and the methodology described here can be used for mapping Mxr1p-binding sites in other methanol-inducible promoters of P. pastoris. Copyright (C) 2010 John Wiley & Sons, Ltd.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Knowledge of protein-ligand interactions is essential to understand several biological processes and important for applications ranging from understanding protein function to drug discovery and protein engineering. Here, we describe an algorithm for the comparison of three-dimensional ligand-binding sites in protein structures. A previously described algorithm, PocketMatch (version 1.0) is optimised, expanded, and MPI-enabled for parallel execution. PocketMatch (version 2.0) rapidly quantifies binding-site similarity based on structural descriptors such as residue nature and interatomic distances. Atomic-scale alignments may also be obtained from amino acid residue pairings generated. It allows an end-user to compute database-wide, all-to-all comparisons in a matter of hours. The use of our algorithm on a sample dataset, performance-analysis, and annotated source code is also included.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

From the molecular mechanism of antagonist unbinding in the ß(1) and ß(2) adrenoceptors investigated by steered molecular dynamics, we attempt to provide further possibilities of ligand subtype and subspecies selectivity. We have simulated unbinding of ß(1) -selective Esmolol and ß(2) -selective ICI-118551 from both receptors to the extracellular environment and found distinct molecular features of unbinding. By calculating work profiles, we show different preference in antagonist unbinding pathways between the receptors, in particular, perpendicular to the membrane pathway is favourable in the ß(1) adrenoceptor, whereas the lateral pathway involving helices 5, 6 and 7 is preferable in the ß(2) adrenoceptor. The estimated free energy change of unbinding based on the preferable pathway correlates with the experimental ligand selectivity. We then show that the non-conserved K347 (6.58) appears to facilitate in guiding Esmolol to the extracellular surface via hydrogen bonds in the ß(1) adrenoceptor. In contrast, hydrophobic and aromatic interactions dominate in driving ICI-118551 through the easiest pathway in the ß(2) adrenoceptor. We show how our study can stimulate design of selective antagonists and discuss other possible molecular reasons of ligand selectivity, involving sequential binding of agonists and glycosylation of the receptor extracellular surface. © 2012 John Wiley & Sons A/S.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Dissertation presented to obtain the Doutoramento (Ph.D.) degree in Biochemistry at the Instituto de Tecnologia Qu mica e Biol ogica da Universidade Nova de Lisboa

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Voltage-dependent anion channels (VDAC) are pore-forming proteins found in the outer mitochondrial membrane of eukaryotes. VDACs are known to play an essential role in cellular metabolism and in early stages of apoptosis. In mammals, three VDAC isoforms have been identified. A proteomic approach was exploited to study the expression of VDAC isoforms in rat, bovine, and chicken brain mitochondria. Given the importance of mitochondrially bound hexokinase in regulation of aerobic glycolysis in brain, we studied the possibility that differences in the relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of type A/type B hexokinase binding sites on brain mitochondria. The spots were characterized, and the signal intensities among spots were compared. VDAC1 was the most abundantly expressed of the three isoforms. Moreover the expression of VDAC1 plus VDAC2 was significantly higher in bovine than in rat brain. Chicken brain mitochondria showed the highest VDAC1 expression and the lowest of VDAC2. Bovine brain mitochondria had the highest VDAC2 levels. We concluded that the nature of hexokinase binding site is not determined by the expression of a single VDAC isoform.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

We examined the effect of Angiotensin II (Ang II) on the interaction between the Ca(2+)/CaM complex and hNHE1. Considering that calmodulin binds to NHE1 at two sites (A and B), amino acids at both sites were modified and two mutants were constructed: SA(1K3R/4E) and SB(1K3R/4E). Wild type and mutants were transfected into PS120 cells and their activity was examined by H(+) flux (J(H+)). The basal J(H+) of wild type was 4.71 +/- 0.57 (mM/min), and it was similar in both mutants. However, the mutations partially impaired the binding of CaM to hNHE1. Ang II (10(-12) and 10(-9) M) increased the J(H+) in wild type and SB. Ang II (10(-6) M) increased this parameter only in SA. Ang II (10(-9) M) maintained the expression of calmodulin in wild type or mutants, and Ang II (10(-6) M) decreased it in wild type or SA, but not in SB. Dimethyl-Bapta-AM (10(-7) M), a calcium chelator, suppressed the effect of Ang II (10(-9) M) in wild type. With Ang II (10(-6) M), Bapta failed to affect wild type or SA, but it increased the J(H+) in SB. W13 or calmidazolium chloride (10(-5) M), two distinct calmodulin inhibitors, decreased the effect of Ang II (10(-9) M) in wild type or SB. With Ang II (10(-6) M), W13 or calmidazolium chloride decreased the J(H+) in wild type or SA and increased it in SB. Thus, with Ang II (10(-12) and 10(-9) M), site A seems to be responsible for the stimulation of hNHE1 and with Ang II (10(-6) M), site B is important to maintain its basal activity. Copyright (C) 2010 S. Karger AG, Basel

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Human transthyretin (TTR) is a homotetrameric protein involved in several amyloidoses. Zn(2+) enhances TTR aggregation in vitro, and is a component of ex vivo TTR amyloid fibrils. We report the first crystal structure of human TTR in complex with Zn(2+) at pH 4.6-7.5. All four structures reveal three tetra-coordinated Zn(2+)-binding sites (ZBS 1-3) per monomer, plus a fourth site (ZBS 4) involving amino acid residues from a symmetry-related tetramer that is not visible in solution by NMR.Zn(2+) binding perturbs loop E-alpha-helix-loop F, the region involved in holo-retinol-binding protein (holo-RBP) recognition, mainly at acidic pH; TTR affinity for holo-RBP decreases similar to 5-fold in the presence of Zn(2+). Interestingly, this same region is disrupted in the crystal structure of the amyloidogenic intermediate of TTR formed at acidic pH in the absence of Zn(2+). HNCO and HNCA experiments performed in solution at pH 7.5 revealed that upon Zn(2+) binding, although the alpha-helix persists, there are perturbations in the resonances of the residues that flank this region, suggesting an increase in structural flexibility. While stability of the monomer of TTR decreases in the presence of Zn(2+), which is consistent with the tertiary structural perturbation provoked by Zn(2+) binding, tetramer stability is only marginally affected by Zn(2+). These data highlight structural and functional roles of Zn(2+) in TTR-related amyloidoses, as well as in holo-RBP recognition and vitamin A homeostasis.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Mutants of each of the four divalent cation binding sites of chicken skeletal muscle troponin C (TnC) were constructed using site directed mutagenesis to convert Asp to Ala at the first coordinating position in each site. With a view to evaluating the importance of site-site interactions both within and between the N- and C-terminal domains, in this study the mutants are examined for their ability to associate with other components of the troponin-tropomyosin regulatory complex and to regulate thin filaments. The functional effects of each mutation in reconstitution assays are largely confined to the domain in which it occurs, where the unmutated site is unable to compensate for the defect, Thus the mutants of sites I and II bind to the regulatory complex but are impaired in ability to regulate tension and actomyosin ATPase activity, whereas the mutants of sites III and IV regulate activity but are unable to remain bound to thin filaments unless Ca2+ is present. When all four sites are intact, free Mg2+ causes a 50-60-fold increase in TnC's affinity for the other components of the regulatory complex, allowing it to attach firmly to thin filaments. Calcium can replace Mg2+ at a concentration ratio of 1:5000, and at this ratio the Ca2 . TnC complex is more tightly bound to the filaments than the Mg2 . TnC form, In the C-terminal mutants, higher concentrations of Ca2+ (above tension threshold) are required to effect this transformation than in the recombinant wild-type protein, suggesting that the mutants reveal an attachment mediated by Ca2+ in the N-domain sites.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

We investigated modulation by ATP, Mg2+, Na+, K+ and NH4 (+) and inhibition by ouabain of (Na+,K+)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, . (Na+,K+)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP ( (M) = 0.09 +/- A 0.01 mmol L-1) of the decapodid III (Na+,K+)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na+,K+)-ATPase activity by K+ also revealed a threefold greater affinity for K+ ( (0.5) = 0.91 +/- A 0.04 mmol L-1) in decapodid III than in other stages; NH4 (+) had no modulatory effect. The affinity for Na+ ( (0.5) = 13.2 +/- A 0.6 mmol L-1) of zoea I (Na+,K+)-ATPase was fourfold less than other stages. Modulation by Na+, Mg2+ and NH4 (+) obeyed cooperative kinetics, while K+ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg2+ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg2+-stimulated ATPases other than (Na+,K+)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na+-ATPase may be involved in the ontogeny of osmoregulation in larval The NH4 (+)-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

Relevância:

100.00% 100.00%

Publicador:

Resumo:

Suramin is a polysulphonated naphthylurea with inhibitory activity against the human secreted group IIA phospholipase A(2) (hsPLA2GIIA), and we have investigated suramin binding to recombinant hsPLA2GIIA using site-directed mutagenesis and molecular dynamics (MD) simulations. The changes in suramin binding affinity of 13 cationic residue mutants of the hsPLA2GIIA was strongly correlated with alterations in the inhibition of membrane damaging activity of the protein. Suramin binding to hsPLA2GIIA was also studied by MD simulations, which demonstrated that altered intermolecular potential energy of the suramin/mutant complexes was a reliable indicator of affinity change. Although residues in the C-terminal region play a major role in the stabilization of the hsPLA2GIIA/suramin complex, attractive and repulsive hydrophobic and electrostatic interactions with residues throughout the protein together with the adoption of a bent suramin conformation, all contribute to the stability of the complex. Analysis of the h5PLA2GIIA/suramin interactions allows the prediction of the properties of suramin analogues with improved binding and higher affinities which may be candidates for novel phospholipase A(2) inhibitors. (C) 2012 Elsevier Inc. All rights reserved.