Double-receptor sandwich supramolecule sensing method for the determination of ATP based on uranyl-salophen complex and aptamer

In this study, we report a double-receptor sandwich supramolecule sensing method for the determination of adenosine triphosphate (ATP). One receptor is a uranyl-salophen complex which can bind the triphosphate group in ATP selectively, and another is an anti-adenosine aptamer which is a single-stranded oligonucleotide and can recognize the adenosine group in ATP specifically. The uranyl-salophen complex was immobilized on the surface of amino-silica gel particles and used as the solid phase receptor of ATP. The anti-adenosine aptamer was labeled with a fluorescent group and used as the labeled receptor of ATP. In the procedure of ATP detection, ATP was first combined with the solid phase receptor and then conjugated with the labeled receptor to form a sandwich-type supramolecule. The conditions of fabricating solid phase receptor and the influence of manifold variables on the determination were studied. The experimental results demonstrate that the method has a number of advantages such as high selectivity, high sensitivity, good stability and low cost. Under optimal conditions, the linear range for detection of ATP is 0.2-5.0 nmol/mL with a detection limit of 0.037 nmol/mL. The proposed method was successfully applied for the determination of ATP in real samples with the recoveries of 96.8-103.3%.     

鼠粒细胞集落刺激因子受体的纯化及鉴定

粒细胞集落刺激因子受体(G-CSFR)在鼠NFS-60细胞中有较高的含量,通过对NFS-60细胞的大规模培养,用CHAPS及超速离心抽提G-CSFR, 经G-CSF亲和层析纯化获得G-CSFR, 采用ABC-ELISA进行鉴定.     

Characterization of the rat liver glucocorticoid receptor purified by DNA-cellulose and ligand affinity chromatography

Two rapid and high yield purification methods for the rat liver glucocorticoid receptor based on differential DNA affinity (method A) and ligand affinity (method B) chromatography are described. In method A, the amount of receptor in rat liver cytosol that can be activated and subsequently eluted from a DNA-cellulose column has been increased to 80% by introducing a second heat activation step. Using this method, 1.5 nmol of 25% pure glucocorticoid receptor can be routinely obtained per day from 15-20 rat livers. Method B yields about 2.2 nmol of 60% pure receptor with an overall yield of congruent to 60%. The quality of these purifications has been controlled by affinity labeling. In each case, more than 95% of purified binding activity represented the intact 92,000 +/- 400-Da glucocorticoid receptor polypeptide as shown by sodium dodecyl sulfate-gel electrophoresis and fluorography. No difference in the labeling pattern was observed using either [3H]triamcinolone acetonide (photoaffinity labeling) or [3H]dexamethasone 21-mesylate (electrophilic labeling). The electrophilic labeling step was performed in the cytosol prior to purification by method A to compare the labeled components thus purified with those obtained when the photoaffinity labeling was performed after the purification. Using this approach, distinct breakdown products of the glucocorticoid receptor were revealed, co-purifying during DNA affinity chromatography. Cross-linked receptor obtained by method A has been further purified to homogeneity by preparative sodium dodecyl sulfate-gel electrophoresis and successfully used as immunogen to raise glucocorticoid receptor antibodies in rabbits. These antibodies raised against glucocorticoid receptor, as well as those previously obtained using affinity chromatography-purified receptor, react with the receptor molecules irrespective of their method of purification. Glucocorticoid receptors purified by methods A and B have been analyzed for specific DNA-binding properties by the nitrocellulose filter binding assay.     

Detection of the low density lipoprotein (LDL) receptor on nitrocellulose paper with colloidal gold-LDL conjugates

Gold-low density lipoprotein (LDL) conjugates were used to detect the LDL receptor on nitrocellulose paper. Solubilized rat liver membrane proteins were subjected to electrophoresis and electroblotted onto nitrocellulose paper. The receptor was then detected as a red band (within 10 min) by overlaying with the LDL conjugates. The coloration was prevented by unlabeled LDL, EDTA, and suramin but not by unlabeled HDL3. In the dot blot assay, detection with the colloidal gold-LDL conjugates was as sensitive as both the autoradiographic method with 125I-labeled LDL and the biotinylated LDL method; the estimated limit of detection by scanning densitometry was 1.6 femtomoles of receptor protein. When the coloration obtained with the colloidal gold-LDL conjugates was intensified by photochemical silver staining, down to 200 attomoles of the LDL receptor could be detected. In this assay, the EDTA-sensitive binding of colloidal gold-LDL to solubilized hepatic membrane proteins was 12 times higher for rats treated with 17 alpha-EE than for normal rats. The use of colloidal gold-LDL conjugates is therefore a very easy, safe, inexpensive, fast and sensitive method for the detection of the LDL receptor on nitrocellulose paper. Furthermore, with silver staining and scanning densitometry, the colloidal gold-LDL conjugates could be used in a dot blot assay to quantify tissue and cell LDL receptors down to attomolar levels.     

Interpretation of agonist affinity estimations: the question of distributed receptor states

In the receptor-transducer model of pharmacological agonism, rejection of the traditional assumption that receptor molecules are in vast excess of transducer molecules permits the receptors to become distributed among unbound, bound and complexed states. Under these conditions, agonist affinities are liable to be overestimated when the method of irreversible receptor antagonism is used. Graphical tests have been developed to detect distribution, and these were applied to experimental data from the interaction between 5-HT and phenoxybenzamine on aortic tissue. Significant receptor distribution was not detected by the method. However, in the model it was assumed that there was a linear relation between the concentration of ternary complex and pharmacological effect. If this relation was replaced with a saturable one the effect of receptor distribution would be masked. The implications for pharmacologists and medicinal chemists are discussed.     

An assay procedure for solubilized thyroid hormone receptor: use of Lipidex

A method using Lipidex-1000 is reported for the assay of thyroid hormone receptor activity. The receptor was extracted from rat liver nuclei, incubated with [125I]T3, and applied to 1-ml disposable pipet tip columns containing a small volume of Lipidex. The resin absorbed the free hormone, and the receptor-bound hormone was recovered in the eluate. The method allowed accurate assay of the receptor activity, satisfying a linear relationship between the activity and the receptor protein concentration. The usefulness of this method was demonstrated by determining the pH optimum and the Kd and Bmax of T3 binding by the receptor. The Lipidex column can be used for the preparation of an unoccupied receptor after dissociation of the endogenously bound hormone.     

A method of active conformation search based on active and inactive analogues and its application to allylamine antimycotics

A new program ACSBAIA (Active Conformation Search Based on Active and Inactive Analogues) for determination of the active conformations was developed based on the rationales that specific functional groups of active analogues could reach and interact with the active site of target receptor by means of the change of conformations, but that of inactive analogues could not interact with the active site owing to conformational restriction. The program consisted of 4 sub-programs: conformation sampling system, active conformation constraint system, inactive conformation exclusion system, and activity prediction system. Pharmacophoric conformation of allylamine antimycotics was studied by this method. Activities of 2 analogues were predicted and tested. The results suggested that the method was scientific and practical. The application of this method was not restricted by the three-dimensional structural knowledge of target receptor. In the absence of structural information about the receptor, the method was     

The use of a hydroxylapatite-filter steroid receptor assay method in the study of the modulation of androgen receptor interaction

Receptors for androgen, estrogen, and glucocorticoid can be assayed by hydroxylapatite adsorption of the radioactive steroid-receptor complex and washing of the adducts on membrane filters mounted on a multiple filter holder. The method is economical, very rapid and sensitive. This new receptor assay method was used to study the modulation of androgen receptor of rat ventral prostate by metal ions, thiols, and ligand structure. The interaction of androgen with the naked receptor is inhibited by 10 microM ZnCl2, CdSO4, or CuSO4 but this inhibition is competed by androgen and is reversed by DTT. The androgen-receptor complex is less sensitive to divalent metal ions but Zn2+, at 3 mM, appears to alter the conformation of the receptor and promote the release of androgen. Certain phenanthrene derivatives exhibited striking structural specificities in their ability to compete with radioactive androgen for binding to the prostate receptor. The results suggest that the receptor has binding preference toward individual ring structure in the steroid.     

The rat ovarian lutropin receptor. Purification, hormone binding properties, and subunit composition

The luteinizing hormone/human choriogonadotropin (hCG) receptor from superovulated rat ovary was purified to homogeneity. A novel scheme based on reverse immunoaffinity chromatography using immobilized antibodies to membrane proteins from receptor down-regulated ovary and subsequent two-step affinity purification on hCG-Sepharose was used to isolate homogeneous receptor. The purification method was also compared to an alternate scheme involving lectin affinity chromatography followed by hCG affinity chromatography. The purified receptor obtained by the latter method was heterogeneous and highly aggregated. The hormone binding properties, molecular size, and subunit composition of the purified receptor obtained by either method were identical. The stability of the receptor during and following solubilization was markedly improved by using 20% glycerol. The pure receptor consists of four nonidentical subunits of molecular weight 79,300 (alpha), 66,400 (beta), 55,300 (gamma), and 46,700 (delta) as indicated by polyacrylamide gel electrophoresis under reducing conditions. All receptor subunits generally, but occasionally excepting the alpha-subunit, were specifically labeled with iodinated hCG in membrane and soluble receptor preparations using bifunctional cross-linking agents. Analysis of the cross-linked hormone-receptor complexes under nonreducing conditions showed the molecular mass of the undissociated receptor to be 268,000 daltons. Hormone binding studies demonstrated that the isolated receptor retained all of the specific binding characteristics expected for the luteinizing hormone/hCG receptor. In combination, these results indicate that the functional and structural properties of the receptor were not altered during purification.     

Evaluation of the number of agonist molecules needed to activate a ligand-gated channel from the current rising phase

We propose a new method for calculating the number of agonist binding sites (n) in ligand-gated receptor channels from the initial phase of the current. This method is based on the fact that the relation between the current (I) and its first-time derivative (I') at the beginning of the current reflects the number of transitions that lead to channel opening. We show that, for constant agonist concentration, the above relationship at t --> 0 provides the number of steps leading to channel opening. When the agonist concentration is not constant but rather increases linearly with time, the corresponding value can be obtained using a slightly modified procedure. The analytical results were compared with computer simulations and a good match between the two was obtained. The theoretical procedure was then validated experimentally using the nicotinic receptor, because, for this receptor, the number of binding sites is well established. Indeed, the expected number of two binding sites was obtained. The method was then tested for the quisqualate-type glutamate receptor channel from the opener muscle of crayfish. The number of this receptor's binding sites is not fully resolved. Our results suggest that, for this glutamate receptor as well, two binding sites must be occupied to open the channel.     

Comparison of monoclonal luteinizing hormone (LH) receptor antibody and LH binding sites in vibratome sections of rat ovary by immunohistochemistry

To identify luteinizing hormone (LH) receptors, a monoclonal antibody (MAb) was produced by immunization of Balb/c mice with rat luteal cell membranes. Hybridomas, produced by a method for proteins of low antigenicity, were selected by competition with [125I]-hCG (LH) for luteal membrane binding. Conditions for analysis of LH receptor antibody (IgG2b isotype) binding by immunohistochemistry with an avidin-biotin-peroxidase complex were examined and results compared to localization of bound hCG, to detect receptors. By light microscopy, both bound hCG and the LH receptor antibody were located on luteal cell surfaces. In addition, the LH receptor antibody was associated with luteal cell cytoplasm. Cell surface membrane binding, but not cytoplasmic staining, was reduced in ovaries from rats injected with hCG. By electron microscopy, LH receptor antibody was observed in patches on luteal cell surface membranes and was associated with polysomes, small vesicles, and occasionally with discrete areas of endoplasmic reticulum. Therefore, detection of LH receptors with bound hCG may be limited to receptors found on cell surfaces, while additional LH receptors are revealed by use of a receptor antibody. The cytoplasmic LH receptor may represent stages in the processing of receptor protein. Furthermore, the methodology used in this study should be generally useful for immunohistochemistry with other MAb to receptors.     

Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs.

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.     

Affinity purification of the opioid receptor in NG 108-15 cells using an avidin-biotin system with a novel elution method

Affinity purification of the opioid receptor in NG 108-15 cells was carried out using an affinity resin based on the avidin-biotin interactions, but a new elution method was employed with a radioligand of sub-micromolar concentration. A synthesized biotinyl derivative of leucine-enkephalin has a high affinity for the delta-receptor, but the affinity was lowered 10-fold in the presence of avidin. The new elution method is based on this affinity decrease and resulted in a 100-fold purification over the initial crude materials in the single step. SDS-polyacrylamide gel electrophoresis of the purified receptor revealed three polypeptides of 58, 65 and 71 kDa as possible components of the delta-receptor.     

Purification and characterization of the rat ovarian receptor for luteinizing hormone. Structural studies of subunit interaction

We have purified the luteinizing hormone (LH)/human choriogonadotropin (hCG) receptor by sequential affinity column on wheat germ lectin-Sepharose and hCG-Sepharose. The method was designed to allow also the purification of lactogen receptor from the initial starting material. The purified LH/hCG receptor retained full binding affinity and was identified as a single protein of Mr = 73,000 +/- 3,000 on sodium dodecyl sulfate-gel electrophoresis. Cross-linking studies performed after binding of hCG to the purified LH/hCG receptor indicated that the hCG alpha-subunit undergoes predominant interaction with the receptor molecule. The influence of the beta-subunit in this interaction seems to occur mainly through its association with the alpha-subunit, presumably by conferring specificity to the alpha-subunit for its hormonal interaction with the receptor. The technique described in this study is simple and allows rapid purification of microgram amounts of biologically active receptor suitable for further molecular characterization, microsequencing, and functional reconstitution studies.     

A dot-blot assay for the low density lipoprotein receptor

We describe a new method for detecting the interaction of low density lipoprotein with its receptor using unmodified nitrocellulose as support for membrane protein. The method is specific and sensitive down to 3 micrograms of membrane protein. Unlabeled LDL, but not HDL, competes with 125I-labeled LDL for binding, and binding is abolished by pretreatment of the membranes with pronase and is dependent upon the presence of Ca2+. Furthermore, modification of arginine or lysine residues on LDL abolishes the lipoprotein interaction with the receptor protein supported on the nitrocellulose. When the membranes are solubilized with octyl glucoside, purification steps of the receptor can be directly followed with no interference of the detergent, therefore eliminating the need for its removal. The increased expression of LDL receptors on liver membranes from estradiol-treated rats was also demonstrated. We suggest, therefore, that this method can be used to detect the presence of LDL receptors on minute amounts of membrane protein.     

Purification of the type II insulin-like growth factor receptor from rat placenta

The membrane receptor for insulin-like growth factor II (IGF II) has been purified to near homogeneity from rat placenta by chromatography of crude plasma membranes solubilized in Triton X-100 on agarose-immobilized IGF II. Elution of the IGF II receptor from the matrix at pH 5.0 in the presence of 1.5 M NaCl resulted in a receptor purification of 1100-fold from isolated plasma membranes, or 340-fold from the Triton extract with an average yield of about 50% in five separate purifications. Analysis of 125I-IGF II binding to the solubilized receptor in the Triton extract and in purified form by the method of Scatchard demonstrated no change in receptor affinity (Kd = 0.72 nM). Sodium dodecyl sulfate electrophoresis of the purified receptor showed one major band at Mr = 250,000 with only minor contamination. Affinity labeling of the receptor in isolated placenta membranes and in purified form using 125I-IGF II and the cross-linking agent disuccinimidyl suberate resulted in covalent labeling of only the Mr = 250,000 band. Such labeling was abolished by unlabeled IGF II but was unaffected by insulin, consistent with the previously reported specificity of IGF II receptor (Massague, J., and Czech, M.P. (1982) J. Biol. Chem. 257, 5038-5045). These results establish a one step affinity method for the purification of the type II IGF receptor that is rapid and highly efficient.     

Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis

Recent evidence suggests that the EGF receptor oligomerizes or clusters in cells even in the absence of agonist ligand. To assess the status of EGF receptors in live cells, an EGF receptor fused to eGFP was stably expressed in CHO cells and studied using fluorescence correlation spectroscopy and fluorescent brightness analysis. By modifying FIDA for use in a two-dimensional system with quantal brightnesses, a method was developed to quantify the degree of clustering of the receptors on the cell surface. The analysis demonstrates that under physiological conditions, the EGF receptor exists in a complex equilibrium involving single molecules and clusters of two or more receptors. Acute depletion of cellular cholesterol enhanced EGF receptor clustering whereas cholesterol loading decreased receptor clustering, indicating that receptor aggregation is sensitive to the lipid composition of the membrane.     

ReFlexIn: A Flexible Receptor Protein-Ligand Docking Scheme Evaluated on HIV-1 Protease

For many targets of pharmaceutical importance conformational changes of the receptor protein are relevant during the ligand binding process. A new docking approach, ReFlexIn (Receptor Flexibility by Interpolation), that combines receptor flexibility with the computationally efficient potential grid representation of receptor molecules has been evaluated on the retroviral HIV-1 (Human Immunodeficiency Virus 1) protease system. An approximate inclusion of receptor flexibility is achieved by using interpolation between grid representations of individual receptor conformations. For the retroviral protease the method was tested on an ensemble of protease structures crystallized in the presence of different ligands and on a set of structures obtained from morphing between the unbound and a ligand-bound protease structure. Docking was performed on ligands known to bind to the protease and several non-binders. For the binders the ReFlexIn method yielded in almost all cases ligand placements in similar or closer agreement with experiment than docking to any of the ensemble members without degrading the discrimination with respect to non-binders. The improved docking performance compared to docking to rigid receptors allows for systematic virtual screening applications at very small additional computational cost.     

A method of active conformation search based on active and inactive analogues and its application to allylamine antimycotics

A new program ACSBAIA (Active Conformation Search Based on Active and Inactive Analogues) for determination of the active conformations was developed based on the rationales that specific functional groups of active analogues could reach and interact with the active site of target receptor by means of the change of conformations, but that of inactive analogues could not interact with the active site owing to conformational restriction. The program consisted of 4 sub-programs: conformation sampling system, active conformation constraint system, inactive conformation exclusion system, and activity prediction system. Pharmacophoric conformation of allylamine antimycotics was studied by this method. Activities of 2 analogues were predicted and tested. The results suggested that the method was scientific and practical. The application of this method was not restricted by the three-dimensional structural knowledge of target receptor. In the absence of structural information about the receptor, the method was particularly applicable.     

Sensitive nonradioactive screening method for compounds interacting with alpha7-cholinoreceptor

A sensitive nonradioactive method for detection of substances interacting with the neuronal nicotinic acetylcholine alpha 7-type receptor (AChR) was proposed. The method uses biotinylated alpha-cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with alpha-cobratoxin (CTX) as does the whole receptor. LBED was produced by heterologic expression of a gene fragment of the alpha 7 subunit of AChR from the rat brain in Escherichia coli cells sorbed on wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin-peroxidase complex. The ability of other compounds to interact with alpha 7-AChR was checked according to the degree with which they inhibit Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).