共查询到20条相似文献,搜索用时 15 毫秒
1.
Shimizu A Matsushita T Kondo T Inden Y Kojima T Saito H Hirai M 《The Journal of biological chemistry》2004,279(16):16285-16294
At the site of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion to subendothelial connective tissue through binding to the N-terminal domain of the alpha chain of platelet glycoprotein Ib (GPIbalpha). To elucidate the molecular mechanisms of the binding, we have employed charged-to-alanine scanning mutagenesis of the soluble fragment containing the N-terminal 287 amino acids of GPIbalpha. Sixty-two charged amino acids were changed singly or in small clusters, and 38 mutant constructs were expressed in the supernatant of 293T cells. Each mutant was assayed for binding to several monoclonal antibodies for human GPIbalpha and for ristocetin-induced and botrocetin-induced binding of 125I-labeled human VWF. Mutations at Glu128, Glu172, and Asp175 specifically decreased both ristocetin- and botrocetin-induced VWF binding, suggesting that these sites are important for VWF binding of platelet GPIb. Monoclonal antibody 6D1 inhibited ristocetin- and botrocetin-induced VWF binding, and a mutation at Glu125 specifically reduced the binding to 6D1. In contrast, antibody HPL7 had no effect for VWF binding, and mutant E121A reduced the HPL7 binding. Mutations at His12 and Glu14 decreased the ristocetin-induced VWF binding with normal botrocetin-induced binding. Crystallographic modeling of the VWF-GPIbalpha complex indicated that Glu128 and Asp175 form VWF binding sites; the binding of 6D1 to Glu125 interrupts the VWF binding of Glu128, but HPL7 binding to Glu121 has no effect on VWF binding. Moreover, His12 and Glu14 contact with Glu613 and Arg571 of VWF A1 domain, whose mutations had shown similar phenotype. These findings indicated the novel binding sites required for VWF binding of human GPIbalpha. 相似文献
2.
Previously reported methods for quantifying platelet-activating factor (PAF) binding to rabbit platelet membranes were modified for studies of PAF binding to human platelet membranes. The membranes were prepared by the "glycerol lysis" method and PAF binding was quantified by using polyethylene glycol precipitation to recover membrane-bound PAF. Optimal PAF binding required buffers containing 3 to 10 mm KCl and either 5 to 10 mM MgCl2 or 5 to 10 mM CaCl2. NaCl was not as effective as KCl and concentrations of NaCl greater than 3 mM strongly inhibited PAF binding. Maximal binding occurred after incubation for 60 min at 0 degree C and was reversed by the addition of excess unlabeled PAF. PAF binding was saturable. Scatchard analysis of PAF binding to 50 micrograms of membrane protein revealed 10.3 +/- 1.7 x 10(11) receptors per milligram of membrane protein and the receptors had a Kd of 7.6 +/- 1.9 nM. The calculated receptor number, binding affinity, and specificity of binding are similar to those previously calculated for PAF binding to intact human platelets, suggesting that the membrane binding site for PAF is the PAF receptor. 相似文献
3.
Paromomycin binds specifically to a single type of binding site on the 70-S streptomycin-sensitive Escherichia coli ribosome. This site is different from that of dihydrostreptomycin since paromomycin binds to streptomycin-resistant ribosomes and sine dihydrostreptomycin does not compete for paromomycin binding. Paromomycin binding, unlike dihydrostreptomycin binding, is independent of changes in ribosome concentration but influenced by magnesium ion concentration. Moreover, paromomycin does not bind to the 30-S subunit of the streptomycin-sensitive ribosome, except in the presence of dihydrostreptomycin, which probably induces the conformational changes necessary for a paromomycin binding site. This induction does not occur with streptomycin-resistant ribosomes. Neither antibiotic binds to the 50-S subunit. In general, binding of the one antibiotic increases the number of sites available for binding of the other. Both antibiotics exhibit marked non-specific binding at high antibiotic/ribosome ratios. Competition studies have enabled the classification of other aminoglycosides according to their ability to compete for the paromomycin and dihydrostreptomycin binding sites. Derivatives structurally related to paromomycin compete for its binding, the degree of competition being related to antibacterial activity, but do not compete for dihydrostreptomycin binding; they, on the contrary, increase the number of dihydrostreptomycin binding sites. Neither gentamicin nor kanamycin derivatives, which induce a high level of misreading, nor kasugamycin and spectinomycin, which do not induce misreading, compete for paromomycin or dihydrostreptomycin binding sites. Other sites may be involved in the binding of these aminoglycosides and in inducing misreading. 相似文献
4.
Poland D 《Biopolymers》2001,58(5):477-490
We illustrate a new method for the determination of the complete binding polynomial for nucleic acids based on experimental titration data with respect to ligand concentration. From the binding polynomial, one can then calculate the distribution function for the number of ligands bound at any ligand concentration. The method is based on the use of a finite set of moments of the binding distribution function, which are obtained from the titration curve. Using the maximum-entropy method, the moments are then used to construct good approximations to the binding distribution function. Given the distribution functions at different ligand concentrations, one can calculate all of the coefficients in the binding polynomial no matter how many binding sites a molecule has. Knowledge of the complete binding polynomial in turn yields the thermodynamics of binding. This method gives all of the information that can be obtained from binding isotherms without the assumption of any specific molecular model for the nature of the binding. Examples are given for the binding of Mn(2+) and Mg(2+) to t-RNA and for the binding of Mg(2+) and I(6) to poly-C using literature data. 相似文献
5.
6.
The binding of adenine nucleotides to liver adenylate kinase was dependent on Mg2+ ions. Citric acid enhanced the binding of all metal-chelated radioactive nucleotides and indicated two observable binding sites for Mg3H-ADP and Mg3-ATP and one-half binding site for Mg3H-AMP. Two binding sites of Mg3H-ADP and one binding site for Mg3H-ATP were also observed in the absence of citric acid. Stoichiometric binding of 14C-citric acid to liver adenylate kinase varied with additions of different nucleotides. AMP prevented whereas ADP and ATP enhanced the binding of 14C-citric acid. 相似文献
7.
Our study was designed to examine how components of complex mixtures can
inhibit the binding of other components to receptor sites in the olfactory
system of the spiny lobster Panulirus argus. Biochemical binding assays
were used to study how two- to six-component mixtures inhibit binding of
the radiolabeled odorants taurine, L-glutamate and
adenosine-5'-monophosphate to a tissue fraction rich in dendritic membrane
of olfactory receptor neurons. Our results indicate that binding inhibition
by mixtures can be large and is dependent on the nature of the odorant
ligand and on the concentration and composition of the mixture. The binding
inhibition by mixtures of structurally related components was generally
predicted using a competitive binding model and binding inhibition data for
the individual components. This was not the case for binding inhibition by
most mixtures of structurally unrelated odorants. The binding inhibition
for these mixtures was generally smaller than that for one or more of their
components, indicating that complex binding interactions between components
can reduce their ability to inhibit binding. The magnitude of binding
inhibition was influenced more by the mixture's precise composition than by
the number of components in it, since mixtures with few components were
sometimes more inhibitory than mixtures with more components. These
findings raise the possibility that complex binding interactions between
components of a mixture and their receptors may shape the output of
olfactory receptor neurons to complex mixtures.
相似文献
8.
The binding of the tide compounds to soybean agglutinin was investigated using 13C-NMR spectroscopy. The equilibrium constant for the binding of N-acetyllactosamine was found to be smaller than that obtained for the binding of ovalbumin (1.1 X 10(3) vs. 7.4 X 10(3) M-1). Only two binding sites per lectin tetramer were determined for the binding of ovalbumin, which is half the number of binding sites reported for the binding of small ligands to the lectin. Steric interference between the bulky ovalbumin molecules is believed to be the reason for the observed decrease in the apparent number of binding sites on the lectin. 相似文献
9.
Anomalous insulin-binding activity in the bovine neural retina: a possible mechanism for regulation of receptor binding specificity 总被引:4,自引:0,他引:4
R J Waldbillig G J Chader 《Biochemical and biophysical research communications》1988,151(3):1105-1112
Crude membrane from the bovine neural retina contains one IGF-I and two insulin binding sites. Although both insulin binding sites have a high affinity for insulin (IC50 = 0.1 and 7.0 nM), only one exhibits "classical" specificity and binds insulin with higher affinity than IGF-I. The second insulin binding site is "non-classical" in that it has an equal affinity for IGF-I and insulin. Retinal IGF-I binding exceeds insulin binding by a factor of 10-20. Despite this high level of IGF-I binding it is unlikely that non-classical insulin binding represents insulin binding to an IGF-I receptor because 1) anomalous binding is 30 times greater than that predicted from cross-specificity, 2) low concentrations of unlabeled IGF-I increase IGF-I binding to the IGF-I binding site but do not increase IGF-I binding to the non-classical insulin binding site and 3) the IGF-I receptor's affinity for insulin (and IGF-I) increases greatly during receptor purification. In contrast, the insulin affinity of the non-classical insulin binding site is largely unaffected by this process. Although receptor solubilization and purification had no effect on the insulin receptor's affinity for insulin, it did markedly increase this site's affinity for IGF-I. Thus, the major proportion of purified retinal "insulin receptors" have a higher affinity for IGF-I than insulin. The evidence presented here is consistent with the view that the bovine retina contains one IGF-I and two insulin binding sites and that a detergent-sensitive factor regulates IGF-I affinity of both classes of binding sites. 相似文献
10.
Abstract: Apparent specific binding of [3 H]imipramine to human platelet membranes at high concentrations of imipramine showed deviation from that expected of a single binding site, a result consistent with a low-affinity binding site. The deviation was due to displaceable, saturable binding to the glass fibre filters used in the assays. Imipramine, chloripramine, desipramine, and fluoxetine inhibited binding to filters whereas 5-hydroxytryptamine and ethanol were ineffective. Experimental conditions were developed that eliminated filter binding, allowing assay of high and low-affinity binding to membranes. Failure to correct for filter binding may lead to overestimation of binding parameters, Bmax and KD for high-affinity binding to membranes, and may also be misinterpreted as indicating a low-affinity binding component in both platelet and brain membranes. Low-affinity binding ( KD < 2 μ M ) of imipramine to human platelet membranes was demonstrated and its significance discussed. 相似文献
11.
One of the many factors involved in determining the distribution and metabolism of a compound is the strength of its binding to human serum albumin. While experimental and QSAR approaches for determining binding to albumin exist, various factors limit their ability to provide accurate binding affinity for novel compounds. Thus, to complement the existing tools, we have developed a structure-based model of serum albumin binding. Our approach for predicting binding incorporated the inherent flexibility and promiscuity known to exist for albumin. We found that a weighted combination of the predicted logP and docking score most accurately distinguished between binders and nonbinders. This model was successfully used to predict serum albumin binding in a large test set of therapeutics that had experimental binding data. 相似文献
12.
ATP is required for the binding of precursor proteins to chloroplasts 总被引:30,自引:0,他引:30
L J Olsen S M Theg B R Selman K Keegstra 《The Journal of biological chemistry》1989,264(12):6724-6729
One of the first steps in the transport of nuclear-encoded, cytoplasmically synthesized precursor proteins into chloroplasts is a specific binding interaction between precursor proteins and the surface of the organelle. Although protein translocation into chloroplasts requires ATP hydrolysis, binding is generally thought to be energy independent. A more detailed investigation of precursor binding to the surface of chloroplasts showed that ATP was required for efficient binding. Protein translocation is known to require relatively high levels (1 mM or more) of ATP. As little as 50-100 microM ATP caused significant stimulation of precursor binding over controls with no ATP. Several different precursors were tested and all showed increased binding upon addition of low levels of ATP. Nonhydrolyzable analogs of ATP did not substitute for ATP, indicating that ATP hydrolysis was required for binding. A protonmotive force was not involved in the energy requirement for binding. Other (hydrolyzable) nucleotides could substitute for ATP but were less effective at stimulating binding. Binding was stimulated by ATP generated inside chloroplasts even when an ATP trap was present to destroy external ATP. We conclude that internal ATP is required for stimulation of precursor binding to chloroplasts. 相似文献
13.
D Martel T G Kennedy M N Monier A Psychoyos 《Journal of reproduction and fertility》1985,75(1):265-274
Membrane preparations from endometria of rats in different physiological states (e.g. pseudopregnancy, ovariectomized animals receiving progesterone + oestradiol or oestradiol alone) were studied for [3H]PGF-2 alpha binding by methods which detected PGF-2 alpha binding in ovary preparations and PGE binding in the same endometrial preparations. There was no evidence of high-affinity binding sites for [3H]PGF-2 alpha. Saturable [3H]PGF-2 alpha binding that increased with the onset of uterine sensitivity was detected but this binding does not fulfil all the criteria required for a PGF-2 alpha receptor and is probably due to binding to PG metabolizing enzymes in our preparations, or to binding of [3H]PGF-2 alpha to PGE binding sites. The failure to detect specific PGF-2 alpha binding sites seems to reflect a true absence of these sites in the rat endometrium. 相似文献
14.
J T Venkatraman Y Lefebvre 《Biochemical and biophysical research communications》1987,148(3):1496-1502
Equilibrium binding of T3 to nuclear matrices isolated from male rat liver occurred after incubation for 3h at 20 degrees C. Two binding sites, having KD's of 6 and 95 nM, were revealed by Scatchard analysis. T3 and Triac competed for the binding of [125I]T3 to the high affinity site whereas only T3 competed for binding to the lower affinity site. Reverse T3 (rT3) did not compete for the binding of T3 to either class of binding sites. The binding sites were highly DNAse-sensitive, and less sensitive to protease treatment. The effect of binding of T3 to nuclear matrices by ATP, DTT and EDTA indicated that the sites are dissimilar to previously identified cytosolic binding sites. The higher affinity site resembles the T3 receptor in affinity and thyroid hormone specificity. The second site represents a new class of thyroid hormone binding sites. Its role in the regulation of thyroid hormone action warrants further investigation. 相似文献
15.
T Ezashi T Kato K Wakabayashi Y Kato 《Biochemical and biophysical research communications》1992,188(1):170-176
Factors binding to consensus sequences of the cAMP-responsive element (CRE) and the AP1 factor binding site (AP1) were investigated using porcine anterior pituitary nuclear extracts. Each element showed specific gel mobility shifts. By reciprocal competition for the AP1 and CRE binding, CRE prevented AP1 binding completely. On the other hand, AP1 decreased the CRE binding considerably to 20%, suggesting that approximately 80% of the total CRE binding is due to factors which bind to a common site shared by both CRE and AP1, whereas proteins binding to AP1 alone are absent. Relative binding affinities of AP1 against CRE estimated from the reciprocal competition data were 0.17 for CRE binding and 0.56 for AP1 binding. UV cross-linking experiments showed that CRE and AP1 gave different patterns consisting of different molecular size. Inconsistency of the relative binding affinities and the multiple molecular size of binding factors, cannot be explained simply by the presence of two types of binding factor, common CRE/AP1-binding and specific CRE-binding factors. A more likely explanation is that the CRE/AP1-binding factors alter the dimer form by changing each respective partner to bind CRE and/or AP1. 相似文献
16.
Strong agonists cause platelets to expose a procoagulant surface supporting the assembly of two important coagulation enzyme complexes. Equilibrium binding has determined the density of high affinity saturable factor IXa binding sites to be 500-600 sites/platelet. We have now used flow cytometry to visualize the binding of factor IX and IXa to thrombin- or SFLLRN-activated platelets. Concentrations of these agonists that are half-maximal or maximal in kinetic studies resulted in only a small subpopulation (4-20%) of platelets binding factor IX or IXa with the density of binding sites for factor IX being about half of that for factor IXa, consistent with previous equilibrium binding studies. A small subpopulation (5 +/- 1.5%) of platelets stimulated with either agonist also exposed annexin V binding sites, and this subpopulation of platelets also bound factor IXa. Annexin V decreased factor IXa binding in the presence or absence of factor VIIIa, and factor IXa could also decrease annexin V binding on some platelets indicating a common binding site in agreement with previous studies. All platelets binding factor IXa were positive for glycoprotein IX, at the same glycoprotein IX surface density as seen in platelets negative for factor IXa binding. These studies refine the results from equilibrium binding studies and suggest that, on average, only a small subpopulation (approximately 10%) of PAR 1-stimulated platelets expose approximately 6000 factor IXa binding sites/platelet. 相似文献
17.
Terence A. G. Noctor Irving W. Wainer 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1992,577(2)
A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin—azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective. 相似文献
18.
Characterization of the adenosine deaminase-adenosine deaminase complexing protein binding reaction 总被引:3,自引:0,他引:3
W P Schrader C A West A D Miczek E K Norton 《The Journal of biological chemistry》1990,265(31):19312-19318
Glutaraldehyde-fixed membranes from rabbit kidney cortex were used to characterize binding of monomeric adenosine deaminase to the adenosine deaminase complexing protein. With the use of bovine adenosine deaminase it was shown that enzyme binding is a saturable, high affinity process. The K value for binding of the bovine enzyme was 11 nM. Maximum enzyme binding and rate of binding to a constant amount of membrane did not vary significantly from pH 5.0 to 9.5. Metal ions, with the exception of Hg2+, sulfhydryl reagents, and other proteins had little or a slightly stimulatory effect on maximum binding. Mercuric ion inhibited binding. Using biotinylated bovine adenosine deaminase it was shown that purified rabbit, human, and monkey enzymes compete for binding sites on fixed membranes. The K values for the rabbit and human enzymes were 9 and 6 nM, respectively. Mouse or guinea pig adenosine deaminase did not bind to the membranes or compete with the biotinylated bovine enzyme for binding sites. The retention of characteristics required for binding by enzymes from rabbit, human, monkey, and calf tissues argues for biologic significance of the adenosine deaminase-complexing protein interaction. The basis for the apparent failure of rodent adenosine deaminase to bind to complexing protein remains to be determined. 相似文献
19.
Jerry A. Stitzel Stephen M. Campbell Allan C. Collins Michael J. Marks 《Journal of neurochemistry》1988,50(3):920-928
Two distinct binding sites with properties corresponding to those expected for nicotinic cholinergic receptors can be identified in brain by the specific binding of nicotine (or acetylcholine) and alpha-bungarotoxin. The effects of modification of these binding sites by treatment with the disulfide-reducing agent dithiothreitol were examined in tissue prepared from DBA mouse brains. Treatment with dithiothreitol reduced the binding measured with either ligand, and reoxidization of the disulfides fully restored binding. The effects of dithiothreitol treatment appeared to be due to a reduction in the maximal binding of nicotine and to a decrease in the binding affinity for alpha-bungarotoxin. Agonist affinity for the alpha-bungarotoxin binding site was reduced by treatment with low concentrations of dithiothreitol. The nicotine binding sites remaining after disulfide treatment displayed rates of ligand association and dissociation similar to those of unmodified tissue, but treatment of previously unmodified tissue with dithiothreitol accelerated the rate of nicotine dissociation. After reduction, both binding sites could be selectively alkylated with bromoacetylcholine. The results suggest that both putative nicotinic receptors in brain respond similarly to disulfide reduction and that their responses resemble those known for the nicotinic receptor of electric tissue. 相似文献
20.
Evidence for the Importance of a Carboxyl Group in the Binding of Ligands to the D2 Dopamine Receptor 总被引:2,自引:2,他引:0
A series of group specific modifying reagents were tested for their effects on [3H]spiperone binding to brain D2 dopamine receptors to identify amino acid residues at the binding site of the D2 dopamine receptor that are critical for ligand binding. The dependence of ligand binding to the receptor on the pH of the incubation medium was also examined. N-Acetylimidazole, 5,5'-dithiobis(2-nitrobenzoic acid), 1,2-cyclohexanedione, and acetic anhydride had no specific effect on [3H]spiperone binding, indicating the lack of participation of tyrosine, free sulphydryl, arginine, or primary amino groups in ligand binding to the receptor. N,N'-Dicyclohexylcarbodiimide (DCCD) potently reduced the number of [3H]spiperone binding sites, indicating that a carboxyl group is involved in ligand binding to the receptor. The effects of DCCD could be prevented by prior incubation of the receptor with D2 dopamine receptor selective compounds. The pH-binding profile for [3H]spiperone binding indicated the importance of an ionising group of pKa 5.2 for ligand binding which may be the same carboxyl group. Diethyl pyrocarbonate, the histidine modifying reagent, also inhibited [3H]spiperone binding, reducing the affinity of the receptor for this ligand but the effects were not at the ligand binding site. From the effects of pH changes on ligand binding some evidence was obtained for a second ionising group (pKa 7.0) that specifically affects the binding of substituted benzamide drugs to the receptor. It is concluded that the D2 dopamine receptor binding site contains separate but over-lapping binding regions for antagonists such as spiperone and substituted benzamide drugs. The former region contains an important carboxyl group; the latter region contains another group that may be a second carboxyl group or a histidine. 相似文献