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A theoretical binding model is considered which provides an explanation for the inverse protein concentration dependence observed for a variety of ligands. The model describes the inhibition of binding caused by a highly bound contaminant. The complete binding equation is derived and examined in terms of form, limits, and protein dependence. Furthermore, several approximate relations are derived which are useful for obtaining initial estimates of the model parameters and for a qualitative test of the applicability of the model. It is found that the binding curve may show a characteristic plateau at a saturation equal to the uncontaminated fraction of the protein and that the free ligand concentration at half saturation depends linearly on protein concentration. The practical implications of the present findings are discussed based on an analysis of simulated as well as experimental data. 相似文献
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An incomplete separation of free and acceptor-bound ligand causes underestimation of specific binding even though the determinations are corrected with blank or nonspecific binding values. If the failure of the experimental procedure causes contamination of bound ligand with free ligand, Scatchard plots are linear, though their slopes and abscissa intercepts are different from the true ones. On the other hand, if the ligand-acceptor complex is incompletely recovered, Scatchard plots are curvilinear with downward concavity. These problems can be overcome if the separable fractions of free and bound ligand are measured and suitable corrections are applied.The separation of free and receptor-bound 125I-labeled human growth hormone by a precipitation method is taken as an example of the procedure. 相似文献
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The sensitivity for the determination of lithium by emission in the nitrous oxide-acetylene flame rivals that of graphite furnace atomic absorption and is freer of chemical interference. Details of its application to analysis of organs of small animals forlithium at physiological (normal) levels are presented. Matrix matching and standardization by additions are unnecessary. 相似文献
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A method is described for fitting equations to enzyme kinetic data that requires minimal assumptions about the error structure of the data. The dependence of the variances on the velocities is not assumed, but is deduced from internal evidence in the data. The effect of very bad observations ('outliers') is mitigated by decreasing the weight of observations that give large deviations from the fitted equation. The method works well in a wide range of circumstances when applied to the Michaelis-Menten equation, but it is not limited to this equation. It can be applied to most of the equations in common use for the analysis of steady-state enzyme kinetics. It has been implemented as a computer program that can fit a wide variety of equations with two, three or four parameters and two or three variables. 相似文献
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The phenomenological principles of information theory are used in the analysis of ligand-binding phenomena in biological macromolecules. Information maps are constructed to visualize regions of ligand chemical potential with maximum amount of information and to devise suitable experimental strategies therefrom. Extensive simulation studies and analysis of experimental data also point out the properties of information used as a weighting procedure in nonlinear least-squares analyses. 相似文献
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Purified boveine growth hormone labeled enzymatically with iodine-125 was covalently coupled to cyanogen bromide activated Sepharose 4B gel and to diazotized zirconia-clad glass beads. Under the conditions employed, an average of 0.8 and 7.3 mg of hormone were bound per ml of Sepharose and glass, respectively. When the conjugates were incubated in Krebs-Ringer bicarbonate buffer (pH 7.4), three separate radioactive species were detected in the incubation supernatant by chromatography on Sephadex G-75. The elution volumes of two of the species were identical with those of 125-I-labeled growth hormone and Na-125I controls, while the third component eluted as a moleucle of intermediate size. The rate of release of each species from the solid matrix was linear with time over 4 days and increased with temperature from 4 to 37 degrees. Although significantly less growth hormone was released from glass (0.14%/day) than from Sepharose (0.40%/day) at 37 degrees, active hormone in amounts sufficient to be detectable in a biological assay was nevertheless liberated from the former after as little as 4 hr of incubation. By contrast, the rate of release of 125-Iminus- and the intermediate-size compound from glass was significantly greater than from Sepharose, suggesting that protein bound to glass supports is more susceptible to degradation from exposure to ionizing radiation. 相似文献
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It has been previously shown that complexation of Cu2+ is essential for effective uptake of Cu2+ by brain tissues and that 67Cu complexed to His is taken up by a high affinity and a low affinity saturable process (Hartter, D. E., and Barnea, A. (1988) J. Biol. Chem. 263, 799-805). Using rat hypothalamic tissue slices, we defined the ligand specificity for these two uptake processes. The effectiveness of stereoisomers or methyl (Me) derivatives of His in facilitating 67Cu uptake by the high affinity process was in this decreasing order: L-His = D-His = Me-3-N-His greater than Me-ester-His greater than Me-alpha-N-His greater than or equal to Me-1-N-His. By the low affinity process it was: L-His = D-His = Me-3-N-His = Me-ester-His = Me-alpha-N-His greater than Me-1-N-His. When facilitation of 67Cu uptake by 14 different amino acids was evaluated using copper:ligand (Cu:L) ratios of 1:2,000 (high affinity process) or 1:2 (low affinity process), His stood out as the most effective. However, when [Cu2+] was 0.1 microM and the Cu:L ratio was increased from 1:2,000 to 1:20,000, Ala, Gly, Lys, Ser, or Thr was each as effective as His; when [Cu2+] was 10 microM and the Cu:L ratio was increased from 1:2 to 1:2,000, Gln, Glu, Gly, Lys, or Ser was each superior to His in facilitating 67Cu uptake. Moreover, by comparison to 67Cu uptake at a Cu:L ratio of 1:2, increasing the ratio attenuated (His) or enhanced (Gln, Glu, Gly, Lys, Ser) 67Cu uptake. These results indicate that 1) coordination of Cu2+ with the 1-N-imidazole and the alpha-amino (but not with the carboxyl) is essential for His facilitation of 67Cu uptake, and 2) the amino acid specificity for uptake of complexed Cu2+ is a function of both [Cu2+] and the molar ratio of copper to amino acid. These results are consistent with coordination of Cu2+ with at least three nitrogens being a primary factor facilitating copper uptake by brain tissue. 相似文献
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A M Zharkovski? 《Biulleten' eksperimental'no? biologii i meditsiny》1986,102(9):298-301
In the studies of 3H-diazepam binding to the rat brain membranes it has been shown that insufficiently high concentrations of free ligand might lead to incorrect determination of Bmax. Thus, free ligand concentration in the range of 0.5 to 16 nM (the most often used ones) and low receptor-protein concentrations (0.08 to 0.12 mg/sample) were far from being saturating and therefore could not be applied for the analysis in Scatchard coordinates. In this case Bmax value would be considerably below the true Bmax value. It has been concluded that for the determination of Bmax of 3H-benzodiazepine binding the range of concentrations used should be at least 0.25 to 64 nM. 相似文献
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Christine Tans Frans Dubois Zhi-Duan Zhong Michel Jadot Robert Wattiaux Simone Wattiaux-De Coninck 《Biology of the cell / under the auspices of the European Cell Biology Organization》1994,82(1):45-49
Summary— In the work reported here, we have compared the elimination from the blood, the uptake by the liver and the intracellular distribution of bovine growth hormone, free(Gh) or bound to a monoclonal antibody (GhAb). Results show that: a) the elimination from the blood is more rapid for Gh than for GhAb; b) both molecules are quickly taken up by the liver; c) probably after travelling through endosomes, Gh and GhAb get to lysosomes where they are degraded. However, Gh mostly ends in hepatocyte lysosomes while GhAb is recovered to a large extent in sinusoidal cell lysosomes; and d) binding by isolated hepatocytes is markedly less efficient for GhAb than for Gh. 相似文献
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Graphic presentation and analysis of inhibition data from ligand-binding experiments 总被引:2,自引:0,他引:2
D B Bylund 《Analytical biochemistry》1986,159(1):50-57
One technique for the characterization of receptor subtypes involves measuring the inhibition of the binding of a radioligand which is not subtype selective by agonists or antagonists which are subtype selective. Although such data are routinely calculated using computer programs, it is often useful to have a graphical representation of the data. Until now, a "modified Scatchard" or "Hofstee" plot of the form P = -(P/I)(IC50) + 1 (where P is percentage inhibition and I is the inhibitor concentration) has been used. We describe an alternate plot of the form B = -(B X I)(1/IC50) + B0 (where B is the concentration of bound radioligand and B0 is the concentration of radioligand bound in the absence of the inhibitor). This method has the important advantage that the data need not be calculated as percentage inhibition which eliminates the error involved in the experimentally determined B0 value being included in the other values. 相似文献
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The influence of peptidyl-tRNA on the dissociation of yeast 80 S ribosomes into subunits was studied. For this purpose temperature-sensitive (ts) suppressor strain of yeastSaccharomyces cerevisiae carrying a defect in peptide chain termination was used. It was found that peptidyl-tRNA did not influence the dissociation of ribosomes either at high salt concentration or in the presence of dissociation factor (DF) from yeast. After dissociation of yeast ribosomes in 0.5 M KCl, peptidyl-tRNA remains bound to the 60 S subunit. Some characteristics of the termination process and release of nascent polypeptides from yeast ribosomes are discussed. 相似文献
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Klammt C Perrin MH Maslennikov I Renault L Krupa M Kwiatkowski W Stahlberg H Vale W Choe S 《Protein science : a publication of the Protein Society》2011,20(6):1030-1041
G-protein coupled receptors (GPCRs) constitute the largest family of intercellular signaling molecules and are estimated to be the target of more than 50% of all modern drugs. As with most integral membrane proteins (IMPs), a major bottleneck in the structural and biochemical analysis of GPCRs is their expression by conventional expression systems. Cell-free (CF) expression provides a relatively new and powerful tool for obtaining preparative amounts of IMPs. However, in the case of GPCRs, insufficient homogeneity of the targeted protein is a problem as the in vitro expression is mainly done with detergents, in which aggregation and solubilization difficulties, as well as problems with proper folding of hydrophilic domains, are common. Here, we report that using CF expression with the help of a fructose-based polymer, NV10 polymer (NVoy), we obtained preparative amounts of homogeneous GPCRs from the three GPCR families. We demonstrate that two GPCR B family members, corticotrophin-releasing factor receptors 1 and 2β are not only solubilized in NVoy but also have functional ligand-binding characteristics with different agonists and antagonists in a detergent-free environment as well. Our findings open new possibilities for functional and structural studies of GPCRs and IMPs in general. 相似文献
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Evaluation and propagation of confidence intervals in nonlinear, asymmetrical variance spaces. Analysis of ligand-binding data 总被引:6,自引:2,他引:4
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M L Johnson 《Biophysical journal》1983,44(1):101-106
Two problems that are often overlooked in studies employing nonlinear least-squares techniques for parameter estimation are confidence-interval estimation and propagation. When the parameters are correlated, the variance space and consequently the confidence intervals are nonlinear and asymmetrical. The presented mathematical method for the evaluation of confidence intervals and error propagation addresses these problems. The examples employed to demonstrate these methods include linear least-squares and the nonlinear least-squares analysis of ligand-binding problems, such as hormone receptor interactions and oxygen binding to human hemoglobin. The mathematical procedures have proven very useful for analyzing the molecular mechanism of cooperativity in human hemoglobin (Johnson, M. L., and G. K. Ackers, 1982. Biochemistry 21:201-211). 相似文献
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Summary Sequence-specific assignments for the 1H and 15N backbone resonances of cellular retinoic acid-binding protein (CRABP), with and without the bound ligand, have been obtained. Most of the side-chain resonances of both apo- and holo-CRABP have also been assigned. The assignments have been obtained using two-dimensional homonuclear and heteronuclear NMR data, and three-dimensional 1H-15N TOCSY-HMQC and NOESY-HMQC experiments. The secondary structure, deduced from nuclear Overhauser effects, amide H/D exchange rates and H chemical shifts, is analogous in both forms of the protein and is completely consistent with a model of CRABP that had been constructed by homology with the crystal structure of myelin P2 protein [Zhang et al. (1992) Protein Struct. Funct. Genet., 13, 87–99]. This model comprises two five-stranded -sheets that form a sandwich or -clam structure, and a short N-terminal helix-turn-helix motif that closes the binding cavity between the two sheets. Comparison of the data obtained for apo- and holo-CRABP indicates that a region around the C-terminus of the second helix is much more flexible in the apo-protein. Our data provide experimental evidence for the hypothesis that the ligand-binding mechanism of CRABP, and of other homologous proteins that bind hydrophobic ligands in the cytoplasm, involves opening of a portal to allow entry of the ligand into the cavity. 相似文献