Binding of [3H]Imipramine to Human Platelet Membranes with Compensation for Saturable Binding to Filters and Its Implication for Binding Studies with Brain Membranes

Abstract: Apparent specific binding of [³H]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, B_max and K_D 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 ( K_D < 2 μ M ) of imipramine to human platelet membranes was demonstrated and its significance discussed.     

Characterisation of antibody-binding RNAs selected from structurally constrained libraries.

Constrained RNA libraries of limited sequence complexity were constructed and used to select RNA molecules binding to the antigen binding site of an anti-ferritin antibody. The sequences required as primer-binding sites for the selection cycle were designed to form a predictable secondary structure, which greatly facilitated the characterisation of the secondary structures of the selected RNAs. RNA-antibody interactions were studied by real-time interaction analysis to study the dynamic aspects of binding and by circular dichroism spectroscopy to search for conformational changes upon binding. The selected RNAs were analysed with a binding site sequestering assay and were shown to compete with ferritin for binding to the antigen-binding site. The experiments described here indicate that the introduction of strong structural constraints does not have to interfere with the ability to select tightly and specifically binding RNA-molecules.     

Binding of Radioactive Benzylpenicillin to Asporogenous Mutants of Bacillus subtilis During Postexponential Growth

The specific penicillin binding capacity of a postexponential culture of Staphylococcus aureus remains constant, but that of a sporulating Bacillus subtilis culture fluctuates dramatically. An initial decrease in binding capacity during presporulation events is followed by two distinct intervals of enhanced specific binding capacity during the postlogarithmic growth of a sporulating B. subtilis culture. The first peak of enhanced binding occurs during septation, when enzymes for germ cell wall formation are present; and the second peak coincides with cortical biosynthesis. The specific postlogarithmic binding capacities of a number of Spo(-) mutants of B. subtilis were examined to ascertain if specific asporogenous mutations altered the binding pattern observed with the wild-type organism. Four distinct postexponential binding patterns were recognized: (i) a low, constant binding capacity resembling the binding pattern of S. aureus, (ii) a decrease in binding capacity with no subsequent significant peaks, (iii) a decrease in binding capacity followed by a single peak corresponding to the first peak seen with the wild type, (iv) a pattern similar to the wild type. The fourth pattern was observed in a mutant blocked during stage III of sporogenesis which produced forespores that never became refractile. Mutations blocking either one or both periods of enhanced postlogarithmic binding were interspersed throughout a linkage group of spore genes next to lys-2 on the B. subtilis chomosome.     

Kinetic analysis of antibody-antigen interactions at a supported lipid monolayer

Modified phospholipids possessing carboxyl head groups synthesized from phosphatidylethanolamine were incorporated into supported lipid monolayers on top of a thin gold film. A monoclonal antibody was chemically coupled to the modified lipids in these monolayers and the kinetics of antigen binding were determined by surface plasmon resonance. The binding could be analyzed using a conventional 1:1 binding algorithm and the derived kinetic and affinity constants were almost identical to those reported for the same interaction on a dextran hydrogel-based sensor chip. When an antigen was chemically coupled to a modified lipid monolayer, the binding of a monoclonal antibody to this surface was biphasic. A two-step algorithm describing the formation of a 1:2 antibody:antigen complex was developed which accurately described the data and enabled differentiation of the two binding steps. The binding was assayed varying both the concentration of antibody in solution and the density of antigen on the surface. The affinities determined by Scatchard analysis of equilibrium binding levels were similar to those values obtained from an ELISA.     

The binding affinity of human serum albumin and paclitaxel through MMPBSA based on docked complex

The distribution, free concentration and metabolism of drugs can be significantly altered as a result of binding to albumin. At the same time, the conformational of serum albumin was also changed by interaction with low molecular weight drugs. In present work, we first equilibrated HSA in aqueous solution to obtain the solvated-HSA model. Further solvated-HSA was performed molecular docking with paclitaxel to find the binding sites. The two docking HSA-paclitaxel complexes were obtained and further equilibrated by a 12 ns MD simulation. Then, MMPBSA method was used to investigate the binding free energy of them. Finally, we correlated the fluctuations of residues with corresponding changes in the secondary structure by dssp method. Two binding sites of paclitaxel were found on HSA having considered the solvation effect. More hydrogen bonds were formed at site I respected to site II. A larger binding energy for primary binding also indicated that paclitaxel showed higher binding affinity mainly due to the stronger hydrogen bonding interactions. There was a significant difference between the two complexes on structure according to the dssp results. Moreover, structure of the binding sites exhibited more fluctuations after binding paclitaxel compared with other regions. Paclitaxel binding also induced distinct conformational changes in drug binding site even when it was empty and have contributed to a reduced binding capacity of HSA towards adriamycin.     

Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a catalytically incompetent enzyme that allows substrate binding to both the AS and SBS. In the second enzyme, binding to the SBS was impaired by site-directed mutagenesis, whereas in the third enzyme, the AS was blocked using a covalent inhibitor. Both techniques were able to show that AS and SBS have a similar binding affinity.     

Binding of lipoproteins to inert materials revisited with computer-assisted analysis

A number of studies conducted in the last decade showed that saturable ('specific') binding, by itself, does not necessarily imply biological significance. That is, biological ligands were shown to bind to inert materials as well as to biological receptors in a saturable manner. In these studies specific binding was operationally defined as binding that was displaceable by excess concentrations of unlabeled ligand. This method of measuring specific binding is now no longer considered optimal. To investigate whether optimal (computer-assisted) techniques of measuring specific binding--namely, nonlinear least-squares curve fitting of total binding data, with mathematical separation of the total binding into its various components--might ensure biological significance of measured specific binding, we studied the binding of high-density lipoproteins (HDL3) to tissue culture dishes as an example of binding without biological significance. This binding closely followed the paradigm of a ligand interacting with a class of homogeneous, saturable sites and with a class of relatively unsaturable sites, just as it would have if the HDL3 were interacting with an unpurified biological receptor. This finding indicates that computer-assisted analysis, while most accurately describing binding data, nevertheless does not ensure that measured specific binding has biological significance. Saturability is such a nonselective feature of equilibrium binding data that it should probably no longer be considered one of the criteria for deciding whether or not a defined binding site is a receptor.     

The three recombinant domains of human serum albumin. Structural characterization and ligand binding properties.

In an attempt to systematically dissect the ligand binding properties of human serum albumin (HSA), the gene segments encoding each of its three domains were defined based on their conserved homologous structural motifs and separately cloned into a secretion vector for Pichia pastoris. We were able to establish a generally applicable purification protocol based on Cibacron Blue affinity chromatography, suggesting that each of the three domains carries a binding site specific for this ligand. Proteins were characterized by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, gel filtration, N-terminal sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as well as near- and far-UV CD. In addition to the affinity chromatography ligand Cibacron Blue, binding properties toward hemin, warfarin, and diazepam, each of which represents a standard ligand for HSA, respectively, were investigated by the measurement of induced circular dichroism. Clear experimental evidence is provided here for the location of the primary hemin binding site to be on domain I of HSA, and for the primary diazepam binding site to be on domain III. Further, secondary binding sites were found for hemin to be located on domains II and III, and for diazepam on domain I. The warfarin binding site was located primarily on domain II, while on domain I, a secondary binding site and/or parts of the primary binding site were found.     

Thyrotropin (TSH) binding activities in bovine thyroid tissue: possible role of adenosine 3',5'-monophosphate dependent phosphorylation of thyroid plasma membranes in TSH receptor degradation

The relationship between thyroid plasma membrane phosphorylation and thyrotropin (TSH) receptor degradation was investigated by using bovine thyroid tissues. By fractionation of thyroid cytosol (105,000 X g supernatant of thyroid homogenate) in a continuous sucrose density gradient centrifugation, three different TSH binding activities were separated. During the incubation of thyroid plasma membranes, TSH binding activities were spontaneously released in vitro. By fractionation of the fraction containing released TSH binding activities in the same sucrose density gradient centrifugation, three different TSH binding activities were isolated. These peaks of TSH binding activity corresponded to the peaks of TSH binding activity obtained in cytosol fraction. Adenosine 3',5'-monophosphate (cyclic AMP) enhanced the release of TSH binding activities from the plasma membranes in vitro. After fractionation on a sucrose density gradient centrifugation of the supernatant of the plasma membranes which were preincubated with cyclic AMP, three different peaks of TSH binding activity were identified. These peaks corresponded to the peaks obtained in spontaneously released TSH binding activity. In this case, however, the amount of small molecule TSH binding activities was predominant compared to that of large molecule TSH binding activity. During the incubation of the plasma membranes with [r-32P]-ATP and with cyclic AMP, phosphorylated soluble proteins were released. The profile of the phosphorylated soluble proteins in the sucrose density gradient centrifugation showed three different peaks which corresponded to the peaks of binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of imipramine to phospholipid bilayers using radioligand binding assay

Binding of the tricyclic antidepressant imipramine (IMI) to neutral and negatively charged lipid membranes was investigated using a radioligand binding assay combined with centrifugation or filtration. Lipid bilayers were composed of brain phosphatidylcholine (PC) and phosphatidylserine (PS). IMI binding isotherms were measured up to IMI concentration of 0.5 mmol/l. Due to electrostatic attraction, binding between the positively charged IMI and the negatively charged surfaces of PS membranes was augmented compared to binding to neutral PC membranes. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherms were described both by surface partition coefficients and by binding parameters (association constants and binding capacities). It was confirmed that binding of IMI to model membranes is strongly affected by negatively charged phospholipids and that the binding is heterogeneous; in fact, weak surface adsorption and incorporation of the drug into the hydrophobic core of lipid bilayer can be seen and characterized. These results support the hypothesis suggesting that the lipid part of biological membranes plays a role in the mechanism of antidepressant action.     

Analysis of the binding of C-reactive protein to histones and chromatin

C-reactive protein (CRP) is an acute phase serum protein in man which binds to phosphocholine (PC) in a calcium-dependent manner. CRP has been shown to bind to chromatin and nucleosome core particles. However, CRP does not bind to DNA and there is conflicting evidence regarding the binding of CRP to histones. In the present study, binding of CRP to chromatin was confirmed by ELISA using chromatin bound to microtiter wells. When chromatin depleted of histone H1 was used in the same assay, no CRP binding was detected. Similar results were observed using a competitive inhibition ELISA. These results indicate an important role for H1 in the binding of CRP to chromatin. Further studies were done to characterize the binding of CRP to purified individual histones. CRP binding to histones was demonstrated first by blotting. Calf thymus histones were separated on a 15% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with 125I-CRP. CRP bound to H1 and H2A and to a lesser extent to H2B. Non-specific binding to H3 was seen and no binding to H4 was observed. CRP binding to purified individual histones was tested by ELISA. Essentially identical results were seen to those obtained by blotting. CRP binding to the H2A-H2B complex was observed as well as reactivity with trypsin-resistant fragments of H2A, H2B, and H3. By blotting and by ELISA all CRP reactions were blocked by PC and EDTA indicating binding through the calcium-dependent PC-binding site on CRP. These studies further characterize the nature of the binding of CRP to chromatin and histones and show that the presence of H1 on chromatin is required for CRP binding.     

苹果果实细胞质中依赖活体组织的ABA高亲和力特异结合蛋白

将苹果（Malus pumila L.cv.Starkrimon)果肉微粒体和细胞可溶组分在含有^3H-ABA的缓冲介质中分别温育,仅在细胞可溶组分中测到微弱的^3H-ABA结合活性。但是,如何将果肉组织圆片在^3H-ABA缓冲介质中直接温育,经制备亚细胞组分后直接测定,在细胞可溶组分中测到很高的^3H-ABA特异结合活性。果肉圆片用沸水预先热处理使细胞可溶组分中的^3H-ABA结合活性完全丧失,说明ABA结合依赖于组织的活体状态。药理实验证明了ABA结合位点的蛋白质性质,同时证明该蛋白的活性中心具有－SH和丝氨酸基因。ABA结合蛋白对ABA的结合具有可饱和性、可逆性和高亲和力。Scatchard作图证明存在2种ABA结合蛋白,一种具有较高的亲和力,其解离常数（Kd)为2.9mmol/L,另一种亲和力相对较低,其Kd值为71.4nmol/L。用ABA结构相似物进行的竞争实验证明了ABA结合蛋白对配体结合的立体特异性。分析了ABA结合蛋白与ABA结合的时间曲线、pH和温度依赖性。本研究检测到的依赖活体组织的ABA结合蛋白可能是果实发育过程中介导ABA信号的受体。     

Optimum conditions for the binding of insulin to its receptor

The specific binding of insulin to the membranes from lactating mouse mamary gland was studied as a model of hormonereceptor type of binding. The basic ingredients of binding, the concentration of receptor protein and the concentration of labeled insulin were mainly studied. The characteristic changes in specific binding were followed, the adequate regression equation was drawn and the optimum conditions of binding were established for further experiments. The expediency of applying shortened orthogonal plans, regression analysis and graphic conture analysis were proved.     

A comparison of direct and indirect techniques using ferritin-conjugated ligands for the localization of concanavalin A binding sites on isolated hepatocyte plasma membranes

Concanavalin A binding sites have been localized on isolated plasma membranes both by a direct technique involving ferritin-concanavalin A and by an indirect technique in which membranes were treated successively with concanavalin A, rabbit anti-concanavalin A, and ferritin-conjugated sheep anti-rabbit F(ab')2. Binding studies showed that, at saturation, less than 25% of the concanavalin A binding sites were accessible to ferritin-concanavalin A. The decreased binding was apparently related to steric factors, since membranes saturated with the conjugated ligand were able to bind additional concanavalin A, and since the conjugated ligand, once bound to the membrane, caused the same inhibition of the membrane-bound enzyme 5'-nucleotidase as concanavalin A. Nonspecific binding sites accounted for 10% of the total binding of ferritin-concanavalin A and were localized mainly on the cytoplasmic side of the membrane, whereas specific sites were on the external side. The indirect technique, which was expected to increase the binding of ferritin-conjugate to the membrane, resulted in the binding of ferritin to less than 15% of the concanavalin A binding sites, and did not decrease the nonspecific binding.     

Characterization of nuclear factors binding to AT-rich element in the rat p53 promoter

In this study, we identified AT-rich element located at positions -504 to -516 in the rat p53 promoter by DNase I foot printing assay. This region was previously identified as a positive regulatory element in the murine p53 promoter and designated as PBF1 (p53 binding factor 1) binding site. However, the proteins binding to this AT-rich element have not been identified yet. Therefore, we characterized the binding protein by various biochemical methods. First, we confirmed that by the oligonucleotide competition assay, nuclear factors bound to the AT-rich element in a sequence-specific manner. Two binding proteins were identified in southwestern blotting analysis and the molecular masses of the proteins were 60 and 40 kDa, respectively. The proteins were stable to denaturants or ionic strength. Treatment of chelators showed that the binding proteins did not require divalent cation for DNA-binding activity. In addition, the binding proteins were labile to protease treatment. This study showed that 60 and 40 kDa proteins bound to AT-rich element and the physico-chemical properties provided new insights into the binding proteins.     

Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding

Hepatitis C virus (HCV) cell entry involves interaction between the viral envelope glycoprotein E2 and the cell surface receptor CD81. Knowledge of conserved E2 determinants important for successful binding will facilitate development of entry inhibitors designed to block this interaction. Previous studies have assigned the CD81 binding function to a number of discontinuous regions of E2. To better define specific residues involved in receptor binding, a panel of mutants of HCV envelope proteins was generated, where conserved residues within putative CD81 binding regions were sequentially mutated to alanine. Mutant proteins were tested for binding to a panel of monoclonal antibodies and CD81 and for their ability to form noncovalent heterodimers and confer infectivity in the retroviral pseudoparticle (HCVpp) assay. Detection by conformation-sensitive monoclonal antibodies indicated that the mutant proteins were correctly folded. Mutant proteins fell into three groups: those that bound CD81 and conferred HCVpp infectivity, those that abrogated both CD81 binding and HCVpp infectivity, and a final group containing mutants that were able to bind CD81 but were noninfectious in the HCVpp assay. Specific amino acids conserved across all genotypes that were critical for CD81 binding were W420, Y527, W529, G530, and D535. These data significantly increase our understanding of the CD81 receptor-E2 binding process.     

Glutamate receptor binding in juvenile and adult Rana pipiens CNS

Autoradiographic methods were used to map NMDA and quisqualate-sensitive glutamate binding sites in the brain of mature and juvenile Rana pipiens frogs. NMDA and quisqualate-sensitive sites were consistently co-localized in the CNS. The highest glutamate binding occurred in the telencephalon, hypothalamus, and cerebellum. Glutamate binding sites were also specifical neuropil of the optic tectum, consistent with glutamate being the retinal ganglion cell neurotransmitter. The distribution of glutamate binding sites in the brain of juvenile postmate morphic frogs was similar to that in adults. In general, Quis binding increased about twofold in adults compared to juveniles, whereas NMDA binding did not show a comparable developmental increase. To test whether glutamate binding sites are located on retinal axon terminals or on tectal cell dendrites in the optic tectum, juvenile postmetamorphic frogs were enucleated unilaterally, and receptor binding was performed following 1, 3, 7, and 14 days survival. The denervated tectal neuropil showed a delayed decrease in NMDA-and quiequalate-sensitive binding, consistent with the receptors being located on postsynaptic tectal cell dendrites. © 1994 John Wiley & Sons, Inc.     

A ligand-receptor binding assay by receptor immobilization

Using transferrin-transferrin receptor binding as a model of ligand-receptor binding, we have developed a new and simple binding assay for the solubilized receptor. Solubilized membrane proteins containing transferrin receptor were immobilized by covalent binding to beads having chemical reactive epoxide groups, and then 125I-labeled transferrin was added to the beads. Dose-dependent, ligand-specific, and saturable binding of 125I-labeled transferrin to the immobilized membrane proteins were demonstrated and a Scatchard analysis derived affinity of Kd = 1.8 X 10(-9) M was obtained. These results indicate that the immobilization of receptors onto beads may be useful in a simple binding assay of the solubilized receptor.     

Detection and kinetics of mucosal pathogenic bacteria binding with polysaccharides

The detection and kinetics of mucosal pathogenic bacteria binding on polysaccharide ligands were studied using a surface plasmon resonance biosensor. The kinetic model applied curve-fitting to the experimental surface plasmon resonance sensorgrams to evaluate the binding interactions. The kinetic parameters for the mucosal pathogenic bacteria (Pseudomonas aeruginosa, Pseudomonasfluorescens, Serratia marcescens) with the alginate ligand were determined from a kinetic model. In addition, the binding interactions of the mucosal pathogenic bacteria with polysaccharide binding pairs (Pseudomonas aeruginosa/alginate, Streptococcus pneumoniae/pneumococcal polysaccharide, Staphylococcus aureus/pectin) were also compared with their kinetic parameters. The rate constants of association for Pseudomonas aeruginosa with the alginate ligand were higher than those for Pseudomonas fluorescens. Serratia marcescens had no detectable interaction with the alginate ligand. The adhesion affinity of Pseudomonas aeruginosa with alginate was higher than that for the other binding pairs. The binding affinities of the pathogenic bacteria with their own polysaccharide were higher than that of Staphylococcus aureus with pectin. Measuring the contact angle was found to be a feasible method for detecting binding interactions between analytes and ligands.