A new method of iodinating collagens for use in radioimmunoassay.

Purified collagens from a variety of species were iodinated to a high specific activity with the N-hydroxysuccinimide ester of I¹²⁵-labeled p-hydroxyphenyl propionic acid (Bolton-Hunter reagent). Labeling had no effect on the immunoreactivity of the collagen as determined by hemagglutination inhibition. This compound presumably acylates the abundant ?-amino groups of lysyl and hydroxylysyl residues in the collagen molecule. Using this method it is possible to label pepsin-extracted collagen from which the terminal nonhelical extensions containing tyrosine have been cleaved. The application of Bolton-Hunter-labeled collagens to radioimmunoassay of affinity-purfied antibodies against collagen is demonstrated.     

A radioimmunoassay for human insulin receptor correlation between insulin binding and receptor mass.

We have developed a radioimmunoassay for human insulin receptor. Serum from a patient with Type B severe insulin resistance was used as anti-insulin receptor antiserum. Pure human placental insulin receptor was used as reference preparation and 125I labeled pure insulin receptor as trace. The radioimmunoassay was sensitive (limit of detection less than 17 fmol), reproducible (inter and intra-assay coefficients of variation 12.5% and 1.6% respectively) and specific (no crossreactivity with pure placental IGF-1 receptor, insulin and glucagon). The anti-insulin receptor antibody was, however, able to differentiate between insulin receptor from human placenta and from rat liver. To determine the number of insulin binding sites per receptor, we measured insulin binding (by insulin binding assay) and insulin receptor mass (by radioimmunoassay) in solubilized aliquots from 5 human placentas. The molar ratio of insulin binding to receptor mass was 0.86 +/- 0.12 when binding was determined with monoiodinated 125I-Tyr A 14-insulin. It was 1.94 +/- 0.27 when randomly iodinated 125I-insulin was used. In conclusion, using a sensitive, reproducible and specific radioimmunoassay, we have measured insulin receptor mass independent of insulin binding. Our data are most compatible with binding of one insulin molecule per human placental insulin receptor.     

A new graphic method for determining the rate constant for affinity of a ligand for its receptor]

A new method of determination of the equilibrium constant for a ligand binding to acceptor and evaluation of the number of binding sites on the acceptor molecules (or cells) is suggested. The method is simpler, more convenient, and more precise than Klotz's or Scatchard's method.     

Gel-filtration chromatographic method for determining relative binding affinities: rat hepatic estrogen receptor as an example system

We report a gel-filtration-based chromatographic method for separation of specific, nonspecific, and free radioligand in a protein receptor-ligand binding assay for the example of the estrogen receptor ERalpha. This assay affords relative binding affinities (RBAs) without the need for a separate determination of nonspecific binding. The probit method is recommended as the most satisfactory method of evaluating the data. The assay responds to both estrogen agonists and antagonists, mixtures respond additively, and the slopes of the probit plots indicate that all ligands bind to the same site on the estrogen receptor. RBAs obtained with rat and rainbow trout ERalpha were in good agreement, and also with those from other reported assays, consistent with the interspecies conservation of key regions of the ligand binding domain among estrogen receptors.     

Manometric method for determining bicarbonate content in plant leaves.

A method is described for manometric determination of bicarbonate (as little as 0.25 μmoles) in leaves. Conditions such as optimum pH and inactivation of carbonic anhydrase are introduced to stabilize the bicarbonate. Crude leaf extracts are purified with chloroform and Darco G-60. An ionic mixture is separated from macromolecular compounds by elution from a column of Sephadex G-25. Carbon dioxide is released by acidifying the ionic mixture and is determined with a respirometer.     

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 high-throughput fluorescent polarization assay for nuclear receptor binding utilizing crude receptor extract.

A homogenous high-throughput assay has been developed to measure the binding between nuclear receptors and test compounds. This assay applies a fluorescence polarization (FP) detection method using human glucocorticoid receptor (GR) as a model system. Crude receptor extract, which requires no additional purification, is used in the assay. The binding conditions (i.e., DMSO tolerance, temperature, stability, and variability) have been investigated and validated. At the optimized conditions, a signal-to-background ratio of 2:1 and a Z'-factor of 0.7 was achieved in a 384-well format. Several known strong and weak GR ligands have been evaluated in this system. Possible interference of fluorescent compounds and methods to identify false positives are also discussed. This FP-based assay system can potentially be used for many soluble nuclear receptors in high-throughput binding assays.     

A new method for predicting binding free energy between receptor and ligand

A practical method to estimate binding free energy, ΔG_bind, of a given ligand structure to the target receptor has been developed. The method assumes that ΔG_bind is given by the summation of intermolecular interaction energy, ΔG_inter, and partial desolvation energy, ΔG_desolv. ΔG_desolv is calculated from the buried surface area in the complex between the ligand and receptor, based on solvation energy, ΔG_solv, formulated by an equation which can be calibrated with observed values. Then, the method was applied to arabinose-binding protein (ABP) and dihydrofolate reductase (DHFR), after recalibrating the weights for ΔG_inter and each term of ΔG_desolv using observed ΔG_bind data for 29 known ligands to avidin (AV). The usefulness of our method was confirmed by the fact that correlation coefficients between the calculated and observed ΔG_bind's in AV, ABP and DHFR were 0.92, 0.77, and 0.88, whereas the corresponding values obtained by simple force field calculation were 0.79, 0.30, and 0.79, respectively. Further investigations to improve the method and validate the parameters are in progress. Proteins 33:62–73, 1998. © 1998 Wiley-Liss, Inc.     

A new method for determining liver microsomal cholesterol 7alpha-hydroxylase.

A method is described to determine the mass of 7alpha-hydroxy cholesterol synthetized in vitro by liver microsomes without the use of a radioactive substrate.     

A new method for determining co-operativity in neurotransmitter release

It has been accepted for some time that neurotransmitter release exhibits a co-operative dependence on calcium. Here we suggest a new procedure for estimating the co-operativity, based on the early rise of synaptic delay histograms of induced release at low quantal content. Measurements of such histograms at the lobster neuromuscular junction are reported. On the basis of this data, and also of data from the literature for other species, a re-examination is made of the conventional hypothesis that the kinetics of release is primarily determined by the time course of entry and removal of calcium ions. Two major new hypotheses for the nature of co-operativity are discussed, both containing the additional feature that membrane depolarization activates a molecule or complex that only then can bind calcium and induce release. The measurements confirm the hypothesis that the co-operativity arises from the action of several complexes between calcium and a depolarization-activated molecule to initiate the release of a vesicle. The co-operativity exponent is estimated to be between three and five in lobster neuromuscular junction and also in crayfish, macrobrachium, and frog.     

Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.     

A new method for determining the Michaelis constant

A new method is described for evaluating the parameters K(m) and V in the Michaelis-Menten equation, and is illustrated with experimental data from the literature.     

A method for determining binding kinetics applied to thiamine-binding protein

A rapid and simple method for assaying the binding activity of thiamine-binding protein is described. By this assay method, the binding characteristics of rice bran thiamine-binding protein have been evaluated with [14C]thiamine as ligand. Analysis of these data by Scatchard plot resulted in linear plots giving a dissociation constant (Kd) for thiamine of 0.55 microM and a maximum binding (Bmax) of 14.5 pmol of ligand bound/microgram of protein. Thiamine binding to the binding protein was time dependent and reached equilibrium at approximately 20 min. The Kob was 0.18 min-1 and the k1 was 1.25 X 10(5) min-1 M-1. Reversibility of thiamine binding at equilibrium was completed at 60 min with a k2 value of 0.052 min-1. The Kd calculated from the reverse rate constant was 0.42 microM. These results indicated that this binding assay method was substantially reliable and accurate.     

A method for determining DNA and chondrocyte content of articular cartilage

A novel and precise method was devised to study the DNA and chondrocyte content of articular cartilage. It involved the sequential digestion of cartilage matrix with hyaluronidase, trypsin, and collagenase to release the chondrocytes. A direct cell count and DNA assays were then performed on the cells. The concentration of cells was the quotient of the total number of cells and the weight of cartilage used. The DNA content of cartilage is identical to the amount of DNA in the chondrocytes. Our data also confirmed the earlier findings that cell density and DNA content of articular cartilage decreased gradually to a relatively constant level as animals matured to adulthood.     

A high-performance liquid chromatography method for determining transition metal content in proteins

Transition metals are common components of cellular proteins and the detailed study of metalloproteins necessitates the identification and quantification of bound metal ions. Screening for metals is also an informative step in the initial characterization of the numerous unknown and unclassified proteins now coming through the proteomic pipeline. We have developed a high-performance liquid chromatography method for the quantitative determination of the most prevalent biological transition metals: manganese, iron, cobalt, nickel, copper, and zinc. The method is accurate and simple and can be adapted for automated high-throughput studies. The metal analysis involves acid hydrolysis to release the metal ions into solution, followed by ion separation on a mixed-bead ion-exchange column and absorbance detection after postcolumn derivatization with the metallochromic indicator 4-(2-pyridylazo)resorcinol. The potential interferences by common components of protein solutions were investigated. The metal content of a variety of metalloproteins was analyzed and the data were compared to data obtained from inductively coupled plasma-atomic emission spectroscopy. The sensitivity of the assay allows for the detection of 0.1-0.8 nmol, depending on the metal. The amount of protein required is governed by the size of the protein and the fraction of protein with metal bound. For routine analysis 50 microg was used but for many proteins 10 microg would be sufficient. The advantages, disadvantages, and possible applications of this method are discussed.