Improved gel-filtration method for analysis of estrogen receptor binding

A method has been described whereby 100- to 300-mg amounts of breast tumor tissue can be analyzed for cytoplasmic estrogen receptor binding capacity. This procedure differentiates specific from nonspecific estrogen binding. In addition, competitive adsorption of estrogen to Sephadex gel has been measured and true receptor binding capacities evaluated. By submitting the corrected receptor binding data to Scatchard plot analysis, the dissociation constant of the estrogen receptor complex was determined with greater accuracy.     

A liquid chromatographic method for determination of the modification degree of proteins: PEGylated arginase as an example

A liquid chromatographic method was developed to determine the modification degree of PEGylated proteins. This method effectively separated free polyethylene glycol (PEG) from other species in conjugation mixtures on a C4 reversed-phase column using water-acetonitrile gradient elution. Then the concentrations of free PEG were determined according to the integrated area under the curve of its evaporative light scattering detector (ELSD) signal, which was normalized by the PEG standard with similar molecular weights. The actual numbers of PEG attached to proteins, not those of lysines modified, were calculated. This method was performed with PEGylated arginase mixtures as an example and showed clear advantages over 2,4,6-trinitrobenzenesulfonic acid (TNBS) assays.     

Filter assay method for the estrogen receptor protein: Detection of both filled and unfilled estrogen binding sites

New filter assay methods are presented for quantitating both cytoplasmic and nuclear forms of the estrogen receptor protein. These methods exploit the strong adsorption of this protein to glass-fiber filters, which appears to occur without loss of steroid binding affinity. A “direct assay protocol” is described that detects only unfilled (nonliganded) estrogen binding sites. In addition, a convenient “exchange assay protocol” has been developed that detects, in addition, those receptors present whose binding sites have already bound nonradioactive estradiol. For the exchange assay, an extract containing receptor is adsorbed to a filter, which is washed free of unbound steroid and then equilibrated for a prolonged period with an excess volume of buffer containing radioactive estradiol. After brief washing in steroid-free buffer, the radioactivity adsorbed to the filter is measured to determine the amount of receptor present. These assays can be used at either 4 or 23°C, over a broad range of salt concentrations. The background of nonspecific binding is extremely low, due in part to the almost negligible affinity of free estradiol for the glass-fiber support.     

Coumarin-suberoylanilide hydroxamic acid as a fluorescent probe for determining binding affinities and off-rates of histone deacetylase inhibitors

Histone deacetylases (HDACs) are intimately involved in epigenetic regulation and, thus, are one of the key therapeutic targets for cancer, and two HDAC inhibitors, namely suberoylanilide hydroxamic acid (SAHA) and romidepsin, have been recently approved for cancer treatment. Because the screening and detailed characterization of HDAC inhibitors has been time-consuming, we synthesized coumarin-SAHA (c-SAHA) as a fluorescent probe for determining the binding affinities (K_d) and the dissociation off-rates (k_off) of the enzyme–inhibitor complexes. The determination of the above parameters relies on the changes in the fluorescence emission intensity (λ_ex = 325 nm, λ_em = 400 nm) of c-SAHA due to its competitive binding against other HDAC inhibitors, and such determination neither requires employment of polarization accessories nor is dependent on the fluorescence energy transfer from the enzyme’s tryptophan residues to the probe. Our highly sensitive and robust analytical protocol presented here is applicable to most of the HDAC isozymes, and it can be easily adopted in a high-throughput mode for screening the HDAC inhibitors as well as for quantitatively determining their K_d and k_off values.     

Evidence for involvement of tyrosine in estradiol binding by rat uterus estrogen receptor

The possibility of tyrosine involvement in steroid binding by rat uterus estrogen receptor (rER) was investigated. Chemical modification of rER with reagents such as tetranitromethane (TNM) and N-acetylimidazole (NAcI) inhibited estradiol binding. Steroid binding was inhibited to a greater extent at pH 8 than at pH 6, indicating the participation of tyrosine (TNM has increasing affinity for cysteine ove tyrosine at pH 6). Inhibition patterns remained similar for incubations at 0-4 degrees C or 37 degrees C. NAcI inhibited rER steroid binding at 37 degrees C, but not at 0-4 degrees C. Hydroxylamine incubated in the presence of rER and NAcI appeared to reverse this inhibition. Thus, these findings indicate that the phenyl ring and possibly the phenolic hydroxyl of tyrosine are involved in steroid binding of the receptor.     

The relative binding affinities of PDZ partners for CFTR: a biochemical basis for efficient endocytic recycling

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis. Its expression and functional interactions in the apical membrane are regulated by several PDZ (PSD-95, discs large, zonula occludens-1) proteins, which mediate protein-protein interactions, typically by binding C-terminal recognition motifs. In particular, the CFTR-associated ligand (CAL) limits cell-surface levels of the most common disease-associated mutant DeltaF508-CFTR. CAL also mediates degradation of wild-type CFTR, targeting it to lysosomes following endocytosis. Nevertheless, wild-type CFTR survives numerous cycles of uptake and recycling. In doing so, how does it repeatedly avoid CAL-mediated degradation? One mechanism may involve competition between CAL and other PDZ proteins including Na (+)/H (+) exchanger-3 regulatory factors 1 and 2 (NHERF1 and NHERF2), which functionally stabilize cell-surface CFTR. Thus, to understand the biochemical basis of WT-CFTR persistence, we need to know the relative affinities of these partners. However, no quantitative binding data are available for CAL or the individual NHERF2 PDZ domains, and published estimates for the NHERF1 PDZ domains conflict. Here we demonstrate that the affinity of the CAL PDZ domain for the CFTR C-terminus is much weaker than those of NHERF1 and NHERF2 domains, enabling wild-type CFTR to avoid premature entrapment in the lysosomal pathway. At the same time, CAL's affinity is evidently sufficient to capture and degrade more rapidly cycling mutants, such as DeltaF508-CFTR. The relatively weak affinity of the CAL:CFTR interaction may provide a pharmacological window for stabilizing rescued DeltaF508-CFTR in patients with cystic fibrosis.     

A new unextracted-sample radioimmunoassay method for hepatic endogenous nuclear l-tri-iodothyronine content. Validity of its use in determining nuclear receptor binding characteristics

Endogenous l-tri-iodothyronine content in an hepatic nuclear extract was measured by a new unextracted-sample radioimmunoassay method using 8-anilinonaphthalene-1-sulphonic acid to inhibit the l-[¹²⁵I]tri-iodothyronine binding to the nuclear l-tri-iodothyronine receptor within the extract. For this method, the lower sensitivity limit was 3.125 pg/tube, the recovery of added l-tri-iodothyronine was 90–120%, and the between-assay coefficient of variation was 10%. The amount of endogenous l-tri-iodothyronine was 10–40 pg/0.2 ml of hepatic nuclear extract from euthyroid rats, compared with less than 3.125 pg/0.2 ml from thyroidectomized rats. The results obtained by this new method were compared with a Sephadex G-25 column extracted-sample radioimmunoassay method and showed a good agreement. The values for the endogenous l-tri-iodothyronine content were utilized to correct for the l-tri-iodothyronine concentration within the binding assay mixture in order to accurately determine by Scatchard analysis the binding characteristics of the nuclear l-tri-iodothyronine receptor. The validity of the correction for endogeneous l-tri-iodothyronine was demonstrated by using a nuclear extract from a thyroidectomized rat which was preincubated with a small known amount of l-tri-iodothyronine before determining the nuclear l-tri-iodothyronine receptor binding characteristics. When the Scatchard plots were corrected for the preincubated dose, the results obtained were similar to true values, but they were falsely lower when not corrected. It is concluded that the necessity and validity of using endogenous l-tri-iodothyronine corrections in the Scatchard analytical computations of the nuclear l-tri-iodothyronine receptor binding characteristics has been demonstrated, being particularly more important for affinity constant than maximum binding capacity.     

The relative binding affinity of diethylstilbestrol to uterine nuclear estrogen receptor: effect of serum and serum albumin

The relative binding affinity (RBA) of diethylstilbestrol (DES) was determined in nuclear fractions of the rat uterus. DES displayed a two- to threefold greater affinity (RBA = 245 +/- 36) than estradiol (RBA = 100) for nuclear E receptor. The RBA of DES to nuclear E receptor was lowered significantly in the presence of rat serum (43 +/- 1) or human serum (52 +/- 7). Dilution of human serum resulted in a progressive increase in the RBA of DES which approached that observed in the absence of serum. Addition of purified human serum albumin mimicked the decrease in RBA of DES that was observed with serum. The IC50 of estradiol was not changed in the presence of either rat serum or albumin. These data show that DES possesses a greater affinity for nuclear E receptor than estradiol and that serum albumin can modulate DES binding to uterine E receptor.     

Modeling the inhibitor activity and relative binding affinities in enzyme-inhibitor-protein systems: application to developmental regulation in a PG-PGIP system

Within a number of classes of hydrolytic enzymes are certain enzymes whose activity is modulated by a specific inhibitor-protein that binds to the enzyme and forms an inactive complex. One unit of a specific inhibitor-protein activity is often defined as the amount necessary to inhibit one unit of its target enzyme by 50 %. No objective quantitative means is available to determine this point of 50 % inhibition in crude systems such as those encountered during purification. Two models were derived: the first model is based on an irreversible binding approximation, and the second, or equilibrium, model is based on reversible binding. The two models were validated using the inhibition data for the polygalacturonase-polygalacturonase-inhibiting protein (PG-PGIP) system. Theory and experimental results indicate that the first model can be used for inhibitor protein activity determination and the second model can be used for inhibitor protein activity determination as well as for comparison of association constants among enzymes and their inhibitor-proteins from multiple sources. The models were used to identify and further clarify the nature of a differential regulation of expression of polygalacturonase-inhibiting protein in developing cantaloupe fruit. These are the first relations that provide for an objective and quantitative determination of inhibitor-protein activity in both pure and crude systems. Application of these models should prove valuable in gaining insights into regulatory mechanisms and enzyme-inhibitor-protein interactions.     

Novel fluorescence based receptor binding assay method for receptors lacking ligand conjugates with preserved affinity: study on estrogen receptor alpha

In this study a novel general approach is presented that allows for a straightforward design of receptor binding assays. This principle of a receptor binding assay is applied to the estrogen receptor, which is important in the management of breast cancer and for the estimation of the estrogenic potency of chemicals in the environment. The inhibitory concentrations to reduce cell proliferation in 50% of controls for 17-beta-estradiol, 4-hydroxy tamoxifen, and tamoxifen are determined to be 61 nM, 33 nM, and 17 microM, respectively. The measurement time of the nanoparticle based immunoassay format is 3 s. The Z' factor, which is calculated to be 0.89, reflects the excellent assay performance.     

Mapping on the calf estrogen receptor of the binding domain for an antibody interfering with receptor activation

The localization on the calf estrogen receptor of the binding domain for B36 (an IgM antibody which prevents and reverses the effects of receptor activation) has been studied by means of controlled proteolysis of the receptor-estradiol complex using trypsin, chymotrypsin, and papain. We successively determined for intact and proteolyzed receptor-estradiol complex (i) the abilities of estradiol-binding species to aggregate in low salt medium, to bind to nonspecific DNA absorbed onto cellulose, and to interact with B36 antibody in sucrose gradients; (ii) the hydrodynamic properties of estradiol-binding species, by gel permeation chromatography and sucrose gradient centrifugation in high salt media and (iii) the molecular weights of B36-reactive species, by immunoblot analysis. Three tryptic receptor fragments of Mr 36,000, 34,000, and 33,000 and two chymotryptic fragments of Mr 36,000 and 33,000 included both the hormone- and B36-binding domains but did not interact with DNA, whereas at least two receptor fragments resulting from the action of chymotrypsin and papain bound estradiol with high affinity but interacted neither with DNA nor with B36. Taking into account these results and assuming that structure of the calf estrogen receptor is similar to those of sequenced estrogen receptors (which show a highly conserved organization with considerable homologies in the functional domains), we propose that the B36-binding domain is located either between the DNA- and hormone-binding domains (model I) or at the C-terminal end of the estrogen receptor (model II). The regions that include the main proteolytic cleavage sites of the receptor are also specified, and the abilities of the two models of the calf estrogen receptor to account for the effect of B36 on receptor activation are discussed.     

Ligand-receptor interactions: a new method for determining the binding parameters

A new experimental procedure and new plot coordinates that allow determination of the binding parameters of ligand-acceptor interaction have been proposed. Instead of titration of a constant concentration of receptors with changing concentrations of ligand, as requested by the well-known methods of Klotz and Scatchard, a series of sequential dilutions of the reacting ligand-receptor mixture is suggested. This allows the application of a new coordinate system that transforms the binding isotherms into straight lines. The case of one acceptor with two classes of receptors with different binding constants is also considered briefly, where the correspondent graphs are nonlinear. It is suggested that in some cases this approach can be a simple and convenient substitute of the broadly used methods of Klotz and Scatchard.     

A gel electrophoresis method for determining the relative binding constants of biologically active, intercalating fluorescent stains.

It is possible to correlate the relative binding tightness of a variety of biologically active, intercalating fluorescent stains through the use of a simple gel electrophoresis method. By prebinding the stains to a homologous series of oligodeoxyribonucleotides and subjecting them to electrophoresis, a size determination of the smallest oligomer to which they remain bound may be obtained from that oligomer's fluorescence using short-wavelength ultraviolet irradiation. This method may prove to be quite practical for the preclinical testing of certain drug derivatives used in cancer chemotherapy. An estimate of the relative effectiveness of new derivatives, as compared to previously tested drugs, might be obtained from this type of analysis.     

Towards an automated system for the identification of notifiable pathogens: using as an example.

Simple and rapid identification of pathogen species is crucial to the control of many diseases. Here, James Kay, Andrew Shinn and Christina Sommerville demonstrate that statistical classifiers discriminate a notifiable pathogen Gyrodactylus salaris Malmberg, 1957, a lethal ectoparasite of Atlantic salmon, Salmo salar L., from its benign close relatives.     

Cellular activities of 20K- and 22K-hGH do not necessarily correlate with their binding affinities for rat GH receptor

Even though 20K human growth hormone (20K-hGH) has 3-10% binding affinity for the rat liver and adipose tissue microsomes as compared to 22K-hGH, it was also reported that 20K-hGH has the same potency as 22K-hGH in the hypophysectomized rat weight gain assay. In order to investigate the reason why such controversial data exist, we have studied 20K- and 22K-hGH using the rat GH receptor extracellular domain (rGHR-ECD) and full-length rGHR. When we examined the complex formation of rGHR-ECD with 20K- and 22K-hGH in gel filtration assay, 20K-hGH formed no complex while 22K-hGH formed a 1:1 complex. Next, rGHR cDNA was introduced into Ba/F3 cells and CHO-K1 cells, and stable transfectants (Ba/F3-rGHR and CHO-rGHR) were established. In the proliferation of Ba/F3-rGHR cells, 20K-hGH had 10-fold lower activity than 22K-hGH, which is consistent with their affinities for rGHR. But surprisingly, in the Spi2.1 gene promoter activation in CHO-rGHR cells, 20K- and 22K-hGH had the same activity, which was found not only in stable CHO-rGHR clones but also in CHO-K1 cells transiently expressing rGHR. In conclusion, these results indicate that cellular activities of 20K- and 22K-hGH do not necessarily correlate with their binding affinities for rGHR.