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At least 433 records · Page 24Linked to original sources
PubMed2006

[3H]HEMADO--a novel tritiated agonist selective for the human adenosine A3 receptor.

Adenosine A(3) receptors are promising drug targets for a number of conditions like inflammatory diseases including asthma, ischemic injury or certain types of cancer. Consequently, intense efforts are dedicated to the development of selective A(3) agonists and antagonists. The only tritiated agonist that is available for radioligand binding is the nonselective [(3)H]5'-N-ethylcarboxamidoadenosine ([(3)H]NECA). Based on a recently characterized series of 2-substituted adenosine receptor agonists we developed a tritiated selective A(3) radioligand with high affinity. From this series 2-hexyn-1-yl-N(6)-methyladenosine (HEMADO) with a K(i)-value of 1.1 nM at the human A(3) subtype was chosen. HEMADO is 300-fold selective versus the A(1) subtype, and 1100-fold and more than 25,000-fold selective compared to the adenosine A(2A) and A(2B) receptors, respectively. The tritiated derivative [(3)H]HEMADO exhibited the same affinity as the unlabeled precursor. In concentrations up to 10 nM no specific binding to adenosine A(1), A(2A) or A(2B) receptors was observed confirming the high selectivity for adenosine A(3) receptors. Characterization of [(3)H]HEMADO in radioligand binding studies revealed reversible binding to the human adenosine A(3) subtype. In saturation binding studies for the A(3) subtype a K(D)-value of 1.1 nM was determined. Nonspecific binding at a radioligand concentration of 1 nM amounted to 1-2% of total binding. Competition binding with a panel of adenosine receptor ligands clearly confirmed the correct A(3) pharmacology of the binding site labeled by [(3)H]HEMADO. With [(3)H]HEMADO we present a tritiated agonist with high affinity and A(3)-selectivity and very low nonspecific binding. [(3)H]HEMADO is a useful tool for specific screening for A(3) receptor agonists and antagonists in improved radioligand binding assays with the human subtype.

Adenosine↗
PubMed2004

New class of bacterial phenylalanyl-tRNA synthetase inhibitors with high potency and broad-spectrum activity.

Phenylalanyl (Phe)-tRNA synthetase (Phe-RS) is an essential enzyme which catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNA(Phe)), a key step in protein biosynthesis. Phenyl-thiazolylurea-sulfonamides were identified as a novel class of potent inhibitors of bacterial Phe-RS by high-throughput screening and chemical variation of the screening hit. The compounds inhibit Phe-RS of Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus, with 50% inhibitory concentrations in the nanomolar range. Enzyme kinetic measurements demonstrated that the compounds bind competitively with respect to the natural substrate Phe. All derivatives are highly selective for the bacterial Phe-RS versus the corresponding mammalian cytoplasmic and human mitochondrial enzymes. Phenyl-thiazolylurea-sulfonamides displayed good in vitro activity against Staphylococcus, Streptococcus, Haemophilus, and Moraxella strains, reaching MICs below 1 micro g/ml. The antibacterial activity was partly antagonized by increasing concentrations of Phe in the culture broth in accordance with the competitive binding mode. Further evidence that inhibition of tRNA(Phe) charging is the antibacterial principle of this compound class was obtained by proteome analysis of Bacillus subtilis. Here, the phenyl-thiazolylurea-sulfonamides induced a protein pattern indicative of the stringent response. In addition, an E. coli strain carrying a relA mutation and defective in stringent response was more susceptible than its isogenic relA(+) parent strain. In vivo efficacy was investigated in a murine S. aureus sepsis model and a S. pneumoniae sepsis model in rats. Treatment with the phenyl-thiazolylurea-sulfonamides reduced the bacterial titer in various organs by up to 3 log units, supporting the potential value of Phe-RS as a target in antibacterial therapy.

Animals↗
PubMed2004

Growth hormone alters epidermal growth factor receptor binding affinity via activation of extracellular signal-regulated kinases in 3T3-F442A cells.

Epidermal growth factor receptor (EGFR) is a transmembrane protein that binds EGF in its extracellular domain and initiates signaling via intrinsic tyrosine kinase activity in its cytoplasmic domain. EGFR is important in development, cellular proliferation, and cancer. GH is a critical growthpromoting and metabolic regulatory hormone that binds the GH receptor, thereby engaging various signaling pathways, including ERKs. Prior studies suggest cross-talk between the GH receptor and EGFR signaling systems. Using the GH- and EGF-responsive 3T3-F442A preadipocyte, we previously observed that GH, in addition to causing EGFR tyrosine phosphorylation, also induced EGFR phosphorylation that was detected by PTP101, an antibody reactive with ERK consensus phosphorylation sites. This latter phosphorylation was prevented by pretreatment with MAPK kinase (MEK)1 inhibitors, suggesting ERK pathway dependence. Furthermore, GH cotreatment with EGF markedly slowed EGF-induced EGFR degradation and down-regulation, thereby potentiating EGF-induced EGFR signaling. These effects were also MEK1 dependent and suggested ERK pathway-dependent influence of GH on EGF-induced EGFR postendocytic trafficking and signaling. We now explore the impact of GH on cell surface binding of EGF in 3T3-F442A cells. We found that GH pretreatment caused transient, but substantial, lessening of (125)I-EGF binding. Competitive binding experiments revealed that the decreased binding was primarily due to decreased affinity, rather than a change in the number of EGF binding sites. The effect of GH on EGF binding was concentration dependent and temporally correlated with GH-induced ERK activation and EGFR PTP101-reactive phosphorylation. Blockade of the MEK1/ERK but not the protein kinase C pathway, prevented GH's effects on EGF binding, and our results indicate that the mechanisms of GH- and phorbol-12-myristate-13-acetateinduced inhibition of EGF binding differ substantially. Overall, our findings suggest that GH can modulate both EGF binding kinetics and the EGFR's postbinding signaling itinerary in a MEK1/ERK pathway-dependent fashion.

3T3 Cells↗
PubMed1980

Somatogenic receptors of rat liver: regulation by insulin.

Somatogenic (i.e. GH) receptors have been studied on liver microsomal membranes from male and female rats. Tracer bovine GH was displaced from its binding sites by GHs of various species, but was displaced only weakly by PRLs. Specific bovine GH binding was 3.5-fold higher to female rat liver membranes than to membranes from males. Streptozotocin-induced diabetes significantly reduced binding, by 80% in females and 50% in males, while insulin therapy to normalize weight gain reversed the decrease in binding. Competitive binding curves were consistent with two independent classes of binding site: low affinity sites with K equal to 0.5 nm-1 in both sexes, and high affinity sites with K equal to 12.1 nm-1 in males and 21.4 nm-1 in females (P less than 0.001). The addition of excess ovine PRL caused a substantial loss of high affinity binding with little loss in the low affinity region, suggesting a weak somatogenic role for ovine PRL. In diabetic animals, low affinity sites were unchanged from normal, while high affinity sites were decreased in number, with no change in affinity, and restored on insulin therapy. Serum immunoreactive rat GH levels were the same in normal and diabetic, male and female animals. These studies suggest that the apparent hepatic resistance to GH seen in diabetes when liver somatomedin release is low despite normal serum GH might be explained by the loss of GH receptors in this condition.

Animals↗
PubMed1986

m-Azido-phencyclidine covalently labels the rat brain PCP receptor, a putative K channel.

Phencyclidine (PCP) is a schizophrenomimetic drug of abuse. PCP binds with high affinity (apparent dissociation constant, KD less than 10(-6) M) to rat brain membranes and blocks, selectively, a voltage-gated, noninactivating K channel found in rat brain synaptosomes (presynaptic nerve terminals). Thus, it has been proposed that the high-affinity PCP receptor in brain is this K channel. Consistent with this hypothesis, we now show that several K channel blockers displace 3H-PCP from the rat brain receptor. Additionally, we have used a photolabile analog of PCP, m-azido-PCP (Az-PCP), to identify the brain PCP receptor/putative K channel. In the dark, Az-PCP bound reversibly to 2 classes of sites on rat brain synaptic membranes [KD = 0.14 +/- 0.01 microM (n = 5) for high-affinity binding, and KD = 255 +/- 55 microM for low-affinity binding]. Competitive binding studies between Az-3H-PCP and nonlabeled PCP analogs, and between Az-PCP and several tritiated PCP analogs, indicated that the high-affinity Az-PCP binding site is the high-affinity PCP receptor. Several amino-pyridines (APs) and tetraalkylamines (TAAs), which are known to block K channels in excitable cells, were also found to displace 3H-PCP from its high-affinity binding site on rat brain synaptic membranes. The rank order of potency for displacement of 3H-PCP from this site for the APs was 4-AP approximately equal to 3,4-diAP greater than 2-AP much greater than 3-AP; for the TAAs it was TBA greater than TEA much greater than TMA (the tetra-butyl, ethyl, and methyl amines, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aminopyridine↗
PubMed1997

Two mechanisms in the action of repressor deltaEF1: binding site competition with an activator and active repression.

BACKGROUND: Counteraction between activators and repressors is crucial for the regulation of a number of cell-specific enhancers, where an activator and a repressor are mutually competitive in binding to the same site. DeltaEF1 is a repressor protein of delta1-crystallin minimal enhancer DC5 binding at the CACCT site, and inhibits activator deltaEF3 from binding to the overlapped site. It has two zinc finger clusters N-fin and C-fin, close to N- and C-termini, respectively, and a homeodomain in the middle. deltaEF1 also binds to the E2-box sequence CACCTG, and represses E2-box-dependent enhancers. RESULTS: The mechanism of the repressor action of deltaEF1 was investigated by examining various deletion mutants of deltaEF1 for their activity to repress delta1-crystallin enhancer fragment HN which contained DC5 sequence and an additional activator site. Both zinc finger clusters were found to be essential for DNA binding and repression, but the homeodomain was not. In addition, the NR domain close to the N-terminus was required for full repression. The NR domain showed active repression when fused to the Gal4 DNA binding domain. Active repression by deltaEF1, dependent on the NR domain, was also demonstrated in a situation where the binding sites of deltaEF1 and deltaEF3 were separated. N-fin and C-fin in their isolated forms bind the 5'-(T/C)ACCTG-3' and 5'-(t/C)ACCT-3' sequences, respectively, while the homeodomain showed no DNA binding activity. An analysis of DNA binding of the delta(Int)F form, having both N-fin and C-fin, indicated that a single DNA binding domain is assembled from two zinc finger clusters. CONCLUSION: Two mechanisms are involved in the repressor action of deltaEF1. First, a binding site competition with an activator which depends on the integrity of both zinc finger clusters, and second, an active repression to silence an enhancer which is attributed to the NR domain.

Adenovirus E2 Proteins↗
PubMed1981

[Assay of serum 24,25-dihydroxycholecalciferol by a competitive protein binding method].

24-25(OH)2 D3 is a vitamin D metabolite due to renal 24-hydroxylation of 25OHD3. The metabolite has some important biological functions on the bone and intestine. Its affinity to the D serum binding protein allows a competitive binding protein assay to be carried out as for 25OHD3, following a chromatographic purification step on Sephadex LH 20. Serum levels of the metabolite in 18 control subjects were found to be 2,69 +/- 1,34 ng/ml while 25OHD3 was 17,41 +/- 7,39 ng/ml and the ratio 24-25(OH)2D3 /25OHD3 in the serum was 0,15. The assay of 24-25(OH)2D3 is useful for physiological studies and for identification of vitamin D depletion states.

24,25-Dihydroxyvitamin D 3↗
PubMed2006

Determination of drug binding to plasma proteins using competitive equilibrium binding to dextran-coated charcoal.

A method for determination of drug binding to plasma proteins, which is based on the comparison of drug affinities to plasma proteins and dextran-coated charcoal, is described. The method is free of nonspecific binding feature. The fractions unbound obtained by the charcoal method are in good agreement with values from a traditional ultrafiltration method for various low and highly bound compounds. The method presently requires drug concentrations much less than that of plasma proteins. A possibility of using the method to determine protein binding at an arbitrary drug concentration is discussed. A mechanism of nonspecific binding of drug to ultrafiltration membranes, which yields a good agreement with experimental observations, is suggested.

Adsorption↗
PubMed2000

Characterization of an IL-1 receptor from Asterias forbesi coelomocytes.

The tremendous importance of cytokines to immune defensive systems suggests that they have been conserved through evolution. The existence of interleukin (IL)-1-like molecules in several invertebrate groups substantiates this hypothesis. To characterize further the relationship of invertebrate IL-1-like molecules, we have used competitive binding assays to show that invertebrate coelomocytes of the starfish Asterias forbesi possess an IL-1-specific binding protein. Competitive binding experiments used radiolabeled human IL-1alpha. IL-1 bound specifically to the coelomocytes by a single high-affinity binding site (K(d) = 8.72 x 10(-10)/M). There are approximately 6000 binding sites per cell. The specificity of the receptor was confirmed by demonstrating that, among a group of cytokines and lymphokines tested, only vertebrate IL-1- or echinoderm IL-1-like molecules and the vertebrate IL-1 receptor antagonist inhibit IL-1 binding. Treatment of coelomocytes (labeled with IL-1alpha) with bivalent water-soluble crosslinkers identified a membrane protein of approximately 70 kDa to which IL-1 is specifically crosslinked.

Animals↗
PubMed1986

A murine monoclonal antibody derived from the immunization of human thyroglobulin reacts equally with L-thyroxine and reverse triiodo-L-thyronine and has a unique idiotype.

Murine monoclonal antibody (mAb 16.3.2) to human thyroglobulin which bound equally to various thyroglobulins derived from nine species was obtained from the fusion of C3H/He spleen cells sensitized with normal human thyroglobulin. Characterization of mAb 16.3.2 revealed that both L-thyroxine (T4) and reverse triiodo-L-thyroxine (rT3) were very efficient in the competitive binding inhibition test and that a molar ratio between T4 and rT3 needed for 50% inhibition of binding to immunized thyroglobulin was about 1:1. Further studies on the idiotype of mAb 16.3.2 using both binding and competitive binding inhibition tests showed that mAb 16.3.2 had a unique idiotype not cross-reacting with other monoclonal antibodies to thyroglobulins. Therefore, a possible explanation offered was that mAb 16.3.2 was endowed with a unique idiotype to be regulated by a distinct idiotype network from those of other mAbs.

Animals↗
PubMed1997

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (I). Immuno-dominant and immuno-recessive area.

PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immuno-dominant N-terminus and an immuno-recessive C-terminus.

Amino Acid Sequence↗
PubMed1998

Probing RegA/RNA interactions using electrospray ionization-fourier transform ion cyclotron resonance-mass spectrometry.

The interactions of bacteriophage T4 regA protein, a unique translational regulator, with RNAs of various size and sequence were studied using electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry. Using very gentle interface conditions, regA/RNA complexes with a 1:1 binding stoichiometry were observed for all four target RNAs studied, consistent with solution binding studies. Competitive binding of target RNAs and their degradation products with regA demonstrated that the loss of a single nucleotide resulted in a dramatic change in binding affinity in some cases. Competitive binding of regA with four target RNAs revealed similar relative binding affinity order to that suggested by previous in vitro repression experiments. The use of sustained off-resonance irradiation for collisionally induced dissociation of a regA/RNA complex suggested the potential for directly obtaining information regarding the regA binding domain.

Bacterial Proteins↗
PubMed2003

Direct mass spectrometric determination of the stoichiometry and binding affinity of the complexes between nucleocapsid protein and RNA stem-loop hairpins of the HIV-1 Psi-recognition element.

The formation of noncovalent complexes between the HIV-1 nucleocapsid protein p7 (NC) and RNA hairpins SL2-SL4 of the Psi-recognition element was investigated by direct infusion electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The high resolution afforded by this method provided the unambiguous characterization of the stoichiometry and composition of complexes formed by multiple equilibria in solution. For each hairpin, the formation of a 1:1 complex was found to be the primary binding mode in solutions of intermediate salt content (150 mM ammonium acetate). Binding of multiple units of NC was observed with lower affinity and a maximum stoichiometry matching the limit calculated from the number of nucleotides in the construct and the size of the footprint of NC onto single-stranded nucleic acids, thus implying the defolding of the hairpin three-dimensional (3D) structure. Dissociation constants of 62 +/- 22 nM, 178 +/- 64 nM, and 1.3 +/- 0.5 microM were determined for SL2, SL3-2, and SL4, respectively, which are similar to values obtained by spectroscopic and calorimetric methods with the additional confidence offered by a direct, rather than inferred, knowledge of the binding stoichiometry. Competitive binding experiments carried out in solutions of intermediate ionic strength, which has the effect of weakening the electrostatic interactions in solution, provided a direct way of evaluating the stabilizing contributions of H-bonding and hydrophobic interactions that are more sensitive to the sequence and structural context of the different hairpins. The relative scale of binding affinity obtained in this environment reflects the combination of contributions provided by the different structures of both the tetraloop and the double-stranded stem. The importance of the stem 3D structure in modulating the binding activity was tested by a competitive binding experiment that included the SL3-2 RNA construct, a DNA analogue of SL3 (SL3(DNA)), and a DNA analogue in which all four loop bases were replaced with abasic nucleotides (SL3(abasic)). NC was found to bind the A-type double-stranded stem of SL3-2 RNA at least 30 times more tightly than the B-type helical structure of SL3(DNA). Eliminating the stabilization provided by the interactions with the tetraloop bases made the binding of SL3(abasic) approximately 50 times weaker than that of SL3(DNA).

Base Sequence↗
PubMed1988

Classification of the insulin-like growth factor binding proteins into three distinct categories according to their binding specificities.

Competitive binding experiments with insulin-like growth factor (IGF)-1, IGF-2 and des-(1-3)-IGF-1 have confirmed the interpretation based on limited amino-terminal sequence analysis that at least three types of IGF binding protein occur. In addition to the acid stable subunit of the large serum binding protein which exhibits des-(1-3)-IGF-1 binding only slightly less than IGF-1, the small IGF binding proteins can be separated into two classes based on differences in des-(1-3)-IGF-1 and IGF-2 binding potencies.

Animals↗
PubMed1975

Competitive protein binding assay for methotrexate.

A competitive protein binding assay has been developed for methotrexate based on the tight binding of this drug to Lactobacillus casei dihydrofolate reductase (= tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase; EC 1.5.1.3). Free drug may be separated from that bound to reductase by adsorption with dextran--albumin coated charcoal. Scatchard plot analysis of the enzyme--drug interaction confirmed the presence of a single homogeneous class of binding sites with an association constant Ka of 2.1 X 10(8) M-1. This high affinity binding permits detection of methotrexate in the range of 0.3--30 pmol with a coefficient of variation of 15% or less. The predominant circulating folate, 5-methyl tetrahydrofolate, and the clinically useful rescue agent leucovorin (5-formyl tetrahydropteroyl-glutamic acid) do not interfere with the assay, nor does the methotrexate metabolite 4-amino-4-deoxy-10-methylpteroic acid. Assay of clinical samples, including plasma and cerebrospinal fluid, showed close agreement between the previously described enzyme inhibition assay and the more rapid competitive binding method.

Binding Sites↗
PubMed2002

The ternary complex of cytochrome f and cytochrome c: identification of a second binding site and competition for plastocyanin binding.

The complex of yeast cytochrome c and cytochrome f from the cyanobacterium Phormidium laminosum was investigated by NMR spectroscopy. Chemical shift perturbation analysis reveals that residues around the haem edge of cytochrome c are involved in the complex interface. Binding curves derived from an NMR spectroscopy titration at 10 mM ionic strength indicate that there are two sites for cytochrome c with binding constants of approximately 2 x 10(4) M(-1) and 4 x 10(3) M(-1). A protein docking simulation with NMR-derived constraints identifies two sites, at the front (Site I) and back faces (Site II) of the haem region of cytochrome f. Site I is homologous to the binding site previously determined for the natural cytochrome f partner plastocyanin. Site II may represent the binding site for the Rieske protein in the cytochrome bf complex. Cytochrome c and plastocyanin are shown to compete for binding at Site I. The competition appears to involve electrostatic screening rather than simple steric occlusion of the binding site.

Amides↗
PubMed1991

Actin-caldesmon-myosin-subfragment-1 ternary complex viewed by electron microscopy. Competitive actin binding region for caldesmon and myosin subfragment-1.

An earlier electron microscopic study using different caldesmon forms complexed with actin revealed that the aggregates produced display regular periodic striation after antibody labeling of the 35-kDa caldesmon fragment. This approach provides further evidence that a caldesmon fragment, even as small as 15 kDa, can induce actin filaments to assemble into bundles. The observed difference in the compactness of these structures, depending on the use of the 15-kDa fragment instead of the 35-kDa fragment, suggests the existence of more than one actin-binding site in the caldesmon molecule. In this study, the caldesmon-induced process of F-actin association was investigated in the presence of skeletal myosin subfragment-1, using light-scattering methods, cosedimentation experiment and electron microscopic techniques. We show that the actin-caldesmon association is partially destabilized in the presence of subfragment-1 and this leads to a ternary complex formation. Immunogold labelling of the actin filaments still reveals the presence of caldesmon within this structure. This latter result strengthens the hypothesis that actin has a site(s) able to bind both caldesmon and myosin subfragment-1, as detected by recent NMR observations. This evidence is discussed with respect to the regulatory function of caldesmon during smooth muscle contraction.

Actins↗