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1.
The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a KD of 0.9 nM and koff of 6.6x10−4 s−1, and to selectively inhibit chymase activity with an IC50 value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.  相似文献   

2.
[3H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [3H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (Kd) of 1.5 nM was observed when the [3H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (Kd 20 nM) was observed when [3H] QNB concentration was above 20 nM. Using 10 nM [3H] QNB for binding, the second order association rate constant (k1) of 0.064 nM?1 min?1 and first order dissociation rate constant (k2) of 0.078 min?1(T12 8 min) were observed. k2/k1 = Kd of 1.22 nM is in good agreement with Kd = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [3H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [3H] QNB binding than the muscarinic agonist.  相似文献   

3.
Vascular growth factors, including vascular endothelial growth factor and fibroblast growth factor-2, bind to heparan sulfate proteoglycans in the basement membrane. While this binding, storage, and release system provides a critical model for controlled drug release devices, basement membrane—growth factor binding kinetics have not been fully established. We modified endothelial cell—growth factor binding kinetics protocols for the basement membrane. The basement membrane showed low affinity for fibroblast growth factor-2 (K d = 185.8 nM), with a slow off rate (k off = 0.00338 min?1). However, results were confounded by growth factor binding to tissue culture polystyrene in a manner strikingly similar to basement membrane. Since substrate binding could not be blocked, a binding kinetics based correction technique was developed to account for polystyrene growth factor binding. This method was validated by conducting binding kinetics experiments on bacteriologic plates that exhibit little growth factor binding. This novel method will improve our understanding of cell and protein interaction with the basement membrane in health and disease. They can also further be applied to develop biomimetic drug delivery systems.  相似文献   

4.
Tritium-labeled leukotrienes C4 and D4 (LTC4 and LTD4) bind to membranes from guinea pig lung. Binding properties of the two ligands are almost identical. More than 80% of 3H-LTC4 and 3H- LTD4 binding can be blocked by unlabeled LTC4 (IC50 8 nM versus 3H-LTC4 and 8 nM versus 3H-LTD4), LTD4 (12 nM, 16 nM), LTE4 (40 nM, 98 nM), and the leukotriene antagonist FPL 55712 (14 μM, 11 μM). Binding is reversible (50% dissociation at 65 min for both ligands at 25°). Binding of 3H-LTC4 and 3H-LTD4 is enhanced by divalent cations and inhibited by sodium ions, guanine nucleotides, and EDTA. 3H-LTD4 binds in unaltered form, but 3H-LTC4 appears to bind mostly after conversion to 3H-LTD4. The high affinity, reversibility, and regulation by ions and guanine nucleotides of 3H-LTC4 and 3H-LTD4 binding strongly imply that these binding sites are physiological LTD4 receptors.  相似文献   

5.
The tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by high-temperature solid-state catalytic isotope exchange. It was found that [3H]bestim binds with a high affinity to murine peritoneal macrophages (K d 2.1 ± 0.1 nM) and thymocytes (K d 3.1 ± 0.2 nM), as well as with plasma membranes isolated from these cells (K d 18.6 ± 0.2 and 16.7 ± 0.3 nM, respectively). The specific binding of [3H]bestim to macrophages and thymocytes was inhibited by the unlabeled dipeptide thymogen (L-Glu-L-Trp) (K i 0.9 ± 0.1 and 1.1 ± 0.1 nM, respectively). After treatment with trypsin, macrophages and thymocytes lost the ability to bind [3H]bestim. Bestim in the concentration range of 10?10 to 10?6 M reduced the adenylate cyclase activity in the membranes of murine macrophages and thymocytes.  相似文献   

6.
Abstract

Saffron is the red dried stigmas of Crocus sativus L. flowers and used both as a spice and as a drug in traditional therapeutic. The biological activity of saffron in modern medicine is in development. Its numerous applications as an anti-oxidant and anti-cancer agent are due to its secondary metabolites and their derivatives (safranal, crocins, crocetin, dimethylcrocetin). The aim of this study was to examine the interaction of transfer RNA with safranal, crocetin, and dimethylcrocetin in aqueous solution at physiological conditions. Constant tRNA concentration (6.25 mM) and various drug/tRNA (phosphate) molar ratios of 1/48 to 1/8 were used. FT-IR and UV-Visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants and the effects of drug complexation on the stability and conformation of tRNA duplex. External binding mode was observed for safranal crocetin and dimethylcrocetin, with overall binding constants Ksafranal = 6.8 (± 0.34) × 103 M?1, KCRT = 1.4 (± 0.31) × 104 M?1, and KDMCRT = 3.4 (± 0.30) × 104 M?1. Transfer RNA remains in the A-family structure, upon safranal, crocetin and dimethylcrocetin complexation.  相似文献   

7.
The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of nonopioid β‐endorphin receptor, was labeled with tritium to a specific activity of 29 Ci/mmol. [3H]Octarphin was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from rat adrenal cortex (Kd = 35.7 ± 2.3 nM, Bmax = 41.0 ± 3.6 pmol/mg protein). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to α‐endorphin, γ‐endorphin, [Met5]enkephalin, and [Leu5]enkephalin as well. At the same time, the [3H]octarphin‐specific binding with adrenal cortex membranes was inhibited by unlabeled β‐endorphin (Ki = 32.9 ± 3.8 nM). Octarphin at concentrations of 10?9–10?6 M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, whereas intranasal injection of octarphin at doses of 5 and 20 µg/rat was found to reduce the secretion of corticosterone from the adrenals to the bloodstream. Thus, octarphin decreases the adrenal cortex functional activity through the high affinity binding to nonopioid receptor of β‐endorphin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

8.
Intact human platelets bind cytochalasin B (CB) with a capacity of 100– 120 p mols CB/mg protein or approximately 7 × 104 molecules/cell and dissociation constants (KD) ranging from 2 × 10?8 to 10?6 M. Up to 85% of this saturable binding is displaced by 10?5 M cytochalasin E (CE). This CE-sensitive binding also appears heterogeneous with KD similar to those of the overall binding. The CE-insensitive binding, however, appears as a single component with KD ≌ 4 × 10?7 M. The sedimentable constituents from frozen, thawed, and washed cells also bind CB with KD ranging from 2.4 × 10?8 to 1.5 × 10?6 M and a total capacity of approximately 39 p mols/mg protein which accounts for only 4% of the ligand binding to the intact cell. The major portion (60–80%) of this CB binding is displaceable by 500 mM D-glucose and has a KD of 1.5 × 10?6M, while only 10–15% is CE-sensitive with a KD of 2.4 ± 10?8 M. It is concluded that 95% of the saturable CB binding in platelets is associated with the cytosol of which 80–85% is sensitive to CE and that only 3% of the cellular binding is glucose sensitive, membrane-associated binding. If the CE-sensitive binding associated with the cytosol is entirely to actin, the stoichiometry of this binding is approximately one CB to 30 actin monomers, which is greater by an order of magnitude than that for CB binding to muscle actin.  相似文献   

9.
The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (Kd = 2.6 ± 0.2 nM ). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met5]enkephalin, and [Leu5]enkephalin, as well. The [3H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (Ki = 2.4 ± 0.2 nM ) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (Ki = 2.9 ± 0.2 nM ). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [3H]immunorphin with membranes (Ki = 2.8 ± 0.2 nM ). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

10.
Integrins exhibit reversible changes in their ability to bind ligands and these changes enable transient cell adhesion. We recently showed that leukocyte integrin CR3 (complement receptor type three, CD11b/CD18, αmβ2) may be purified in a form that is either capable or incapable of binding soluble, monomeric ligand and that “inactive” CR3 may be rendered capable of binding ligand by addition of an anti-CR3 mAb known as KIM-127 (Cai and Wright, JBC. 270: 14358, 1995). Here, we demonstrate that active CR3 may be rendered inactive by treatment of immobilized receptor with EDTA. EDTA-treated CR3 failed to bind ligand even in the presence of mM Ca2+ and Mg2+, suggesting that EDTA-treatment caused a change in the receptor that is not readily reversed. EDTA-treated receptor did, however, bind ligand upon addition of KIM-127 plus Mg2+ with an affinity (17.8 ± 4.5 nM) similar to untreated, active receptor (12.5 ± 4.7 nM). EDTA-treated CR3 thus exhibits the properties of inactive CR3, in which the ligand binding site is cryptic but subject to exposure by KIM-127. A candidate for the cryptic ligand binding site is the I-domain, a Mg2+-binding region in the α chain of CR3. We found that monomeric C3bi binds directly to recombinant I-domain in a Mg2+-dependent fashion with an affinity of 300 ± 113 nM. These results thus suggest that CR3 may be inactivated by removing tightly bound divalent cation from a cryptic site in CR3.  相似文献   

11.
The specific binding of the muscarinic cholinergic ligand N-methylscopolamine to human foetal brain has been measured. A level of binding of 64 pmol/g protein was found with a dissociation constant, Kd of 0.27 nM. Values of 0.17 nM min?1 and 0.048 min?1 for the association rate constant, Kon, and the dissociation rate constant Koff respectively, were obtained. The pharmacological properties of the binding site were found to be very similar to those reported for muscarinic receptors from adult mammalian brain except that the binding of pirenzepine and the nicotinic antagonists d-tubocurarine and decamethonium shows differences from that seen in adult brain.  相似文献   

12.
The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable ‘enzyme-product’ complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (Kb) values between 10.23?×?103 and 6.52?×?103 M?1. The binding reactions were enthalpy driven with ΔHb values between ?50.99 and ?46.07 kJ mol?1. From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.  相似文献   

13.
—The major toxin of black widow spider venom, α-latrotoxin, can be iodinated with 125I with hardly any loss in biological activity. The radioactive toxin could bind specifically to a dog cerebral cortex synaptosomal membrane preparation but not to a dog liver plasma membrane preparation. The bound protein could be recovered from the neuronal membrane preparation in an unchanged form. Non-specific binding was only 6–10% of the total binding. The protein nature of the presumed receptor was indicated by the complete inhibition of the binding by either heating the membrane preparation at 70°C or treating the membrane with trypsin. Pre-incubation with 2%β-mercaptoethanol also completely inhibited the binding, while 70% inhibition was observed after pre-treatment with 10m M-EDTA or EGTA. From plots of the equilibrium binding data, it could be ascertained that the binding is non-cooperative, with an apparent equilibrium dissociation constant, K1, of 1.0 nM. Kinetic data gave an apparent association rate constant of 8.2 × 105 M?1 s?1. Dissociation followed a biphasic exponential with rate constants of 1.4 × 10?3 and 5.2 × 10?5s?1 corresponding to half-lives of 8.2 min and 3.7 h. Possible schemes for the binding interaction were proposed. Based on the present results and on previous results which indicated that α-latrotoxin causes the release of all neurotransmitters and a depletion of the synaptic vesicle population in vertebrate synapses, a hypothetical mechanism of the action for the toxin was proposed, involving the binding of the toxin to a membrane protein receptor which interacts with filamentous proteins linking the synaptic vesicles to the axolemma.  相似文献   

14.
A Fulton  G D Burrows 《Life sciences》1980,26(18):1505-1508
The drug concentrations which inhibited 50% of 3H-haloperidol specific binding (the IC50) by striatal receptors of fluphenazine dihydrochloride, 7-hydroxy fluphenazine, chlorpromazine hydrochloride, 7-hydroxy chlorpromazine, pericyazine, and nomifensine were measured: 2.7 nM, 3.3nM, 6.3nM, 4.7nM, 360nM.Fluphenazine sulphoxide and the glucuronide of 7-hydroxychlorpromazine were inactive in competing for binding sites. The IC50 of fluphenazine dihydrochloride, chlorpromazine, and pericyazine followed in the same ranking order as their daily clinical doses as antipsychotics.  相似文献   

15.
Levorphanol inhibits Ca++ binding to synaptic membranes in a dose dependent fashion (0.5–10nM) and induces release of calcium previously bound to membranes. The inhibition is noncompetitive and exhibits a Ki of 9 × 10?9M. Naloxone (10 nM) blocks this inhibition while dextrophan (100nM) is without effects. The Ca++ binding exhibits a high affinity (KD=0.84μM) and a saturable capacity of 350 picomles/mg protein. The binding curve is sigmoid with a Hill coefficient of 2.2, while in the presence of levorphanol (0.5 to 50nM), the curve becomes hyperbolic with a Hill coefficient of 1.1. The data suggest the calcium and opiate receptor binding sites are in close proximity and may be functionally linked through sub-unit interactions.  相似文献   

16.
A tritium-labeled synthetic LKEKK pentapeptide corresponding to the sequences 16–20 of human thymosin-α1 and 131–135 of human interferon-α2 was obtained with a specific activity of 28 Ci/mmol. [3H]LKEKK was found to bind with high affinity (K d 3.7 ± 0.3 nM) to the membranes isolated from epithelial cells of rat small intestinal mucosa. The trypsin treatment of the membranes did not affect the binding, thus supporting the nonprotein nature of the peptide receptor. The binding of the labeled peptide was inhibited by unlabeled thymosin-α1, interferon-α2, and cholera toxin B subunit (K i 4.2 ± 0.4, 3.5 ± 0.3, and 4.7 ± 0.3 nM respectively). The pentapeptide did not affect the adenylate cyclase activity within the concentration range of 1–1000 nM.  相似文献   

17.
The tumorous Drosophila melanogaster blood cell line BII has been studied for evidence for the presence of ecdysteroid receptors. The [3H]ponasterone A (pon A)* used in this study has been extensively purified, and the location of the tritium in the molecule has been partially determined. BII cells do not metabolise ecdysteroids. Intact cells demonstrate a considerable specific uptake of [3H]pon A which is saturable, apparently showing two specific components: a very high affinity component (KD = 0.3 nM) and a high affinity component (KD = 2 nM). The specific binding of [3H]pon A to whole cells is compatible with unlabelled ecdysteroids, but not with mammalian steroid hormones. The association rate constant (ka) for [3H]pon A was determined to be 3 × 107M?1min?1 at 21 °C, while the dissociation rate constant (kd) for the specifically bound [3H]pon A was found to be 4.4 × 10?3/min. Together, the kinetic rate constants yield a value of 0.15 nM for the KD. The receptors have been partially characterised in a cell-free extract prepared by sonification of the cells. The optimum pH for extraction and hormone binding is 8.2. Scatchard plots of binding data indicate that the cell-free extract also contains two high affinity specific binding components (kD = 0.1 nM and KD = 1 nM). The hgih affinity binders are macromolecular, as shown by chromatography on Sephadex G-25, and are susceptible to protease digestion, heat, and treatment with N-ethylmaleimide. Sucrose density centrifugation of the labelled receptor shows one peak at approximately 6S. The stability of the receptor preparation has been studied and conditions have been empirically determined (10% w/v sucrose, 25 mM dithioerthreitol, and 10 mM citrate), whereby the binding capacity of the unlabelled receptor is stable for at least 8 weeks if frozen at ?20°C.  相似文献   

18.
Abstract: The effects of some GABA analogues and some drugs on the binding of [3H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [3H]muscimol binding sites by excess(10?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [3H]muscimol binding was potently inhibited by muscimol itself (IC50, 2.5 nM), GABA (1C50, 43 nM), isoguvacine (IC50, 61 nM), and 3-aminopropanesulphonic acid (IC50, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC50, 800 nM). δ- Aminovaleric acid (IC50, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC50, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC50, 23 μM) and p-proline (IC50, 66 μM) also inhibited [3H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [3H]muscimol binding (IC50≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [3H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.  相似文献   

19.
Cobalt(II)-substituted carboxypeptidase A has been found to reversibly bind N3? and NCO?, but not NCS?, in the pH range 5–10, thus including the pH range of activity of the enzyme. The pH dependence of the anion binding constant is affected by two ionizations, which are assigned as those regulating kcat and KM. The electronic and 1H NMR spectra are consistent with a substantially pseudotetrahedral geometry of the anion derivatives.  相似文献   

20.
The tumor promoter 20-3H-phorbol 12,13-dibutyrate bound in a specific manner to particulate preparations from both whole mouse skin and mouse epidermis. The binding, which was comparable in both whole skin and epidermal preparations, occurred rapidly, was reversible upon addition of non-radioactive ligand and showed high affinity (KD = 2.4 × 10?8 M). The potencies of phorbol esters for inhibiting binding of 3H-PDBu corresponded to their biological and tumor-promoting activities: phorbol 12-myristate 13-acetate, KI = 0.74 nM; phorbol 12,13-didecanoate, KI = 16 nM; phorbol 12,13-dibenzoate, KI = 82 nM; mezerein, KI = 98 nM; phorbol 12,13-diacetate, KI = 3 μM; phorbol 12,13,20-triacetate, KI = 5.6 μM; phorbol 13-acetate, KI = 64 μM. The biologically inactive derivatives phorbol (0.88 mM) and 4α-phorbol 12,13-didecanoate (15 μM) did not inhibit binding. Likewise, 3H-PDBu binding was only weakly inhibited by phorbol-related diterpenes which are highly inflammatory but nonpromoting. These structure-activity relationships suggest that the 3H-PDBu binding activity mediates phorbol ester tumor promotion. 3H-PDBu binding was not inhibited by the nonphorbol promoters examined. Similarly, it was not blocked by compounds which antagonize (dexamethasone acetate, 2 μM; retinoic acid, 10 μM) or mimic (epidermal growth factor, 100 ng/ml; melittin, 25 μg/ml; PGE2, 1 μM) some of the effects of the phorbol esters in vivo or in vitro.  相似文献   

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