Determination of ligand binding affinities for endogenous seven-transmembrane receptors using fluorometric microvolume assay technology.

We have developed a fluorescence-based mix and read method for the quantitative determination of receptor-ligand binding interactions. This method was used to determine IC(50) values for peptide ligands of two endogenous seven-transmembrane receptors that are expressed in cultured human cancer cells. Substance P, neurokinin A, and galanin were labeled with Cy5 and were shown to retain their native binding affinities. The cell-associated fluorescence was quantified using a fluorometric microvolume assay technology (FMAT) scanner that was designed to perform high-throughput screening assays in multiwell plates with no wash steps. The binding of fluorescently labeled substance P and neurokinin A was tested on the human astrocytoma cell line UC11 that expresses endogenous NK(1) receptor. Galanin binding was measured on endogenous galanin type 1 receptors in the Bowes neuroblastoma cell line. IC(50) values were determined for substance P, neurokinin A, and galanin and were found to correspond well with reported values from radioligand binding determinations. To demonstrate FMAT as instrumentation for high-throughput screening, it was utilized to successfully identify individual wells in a 96-well plate in which Cy5-substance P binding in UC11 cells was competed with unlabeled substance P. In addition, we developed a two-color multiplex assay in which cells individually expressing neuropeptide Y and substance P receptors were mixed in the same well. In this assay, the fluorescent ligands substance P and neuropeptide Y bound only to their respective cell types and binding was specifically competed. Therefore, two different seven-transmembrane receptor targets can be tested in one screen to minimize reagent consumption and increase throughput.

Childbirth preparation through hypnosis: the hypnoreflexogenous protocol.

A verbatim protocol for the "Hypnoreflexogenous" method of preparation for childbirth is presented wherein the patient is taught to enter a hypnotic state and then prepared for labor and delivery. The method provides a "conditioned reflex" effect conducive to a positive outcome for labor and delivery by enhancing the patient's sense of readiness and control. Previous applications of the method demonstrate patients have fewer complications, higher frequency of normal and full-term deliveries, and more positive postpartum adjustment. The benefit and ultimate cost effectiveness of the method are discussed.

Automated Ferguson analysis of glycoproteins by capillary electrophoresis using a replaceable sieving matrix.

Obtaining accurate molecular weight estimates for glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been difficult due to the lack of SDS binding by the carbohydrate moieties of the proteins. This leads to lower charge-to-mass ratios for SDS-glycoprotein complexes, resulting in over-estimation of molecular weights by SDS-PAGE. In order to minimize these inaccuracies for proteins with abnormal charge-to-mass ratios, a Ferguson plot may be employed. This application requires the determination of relative mobilities for standard proteins in addition to unknowns at several different gel concentrations. Historically, this technique has not been popular because it requires time-consuming preparation of gels with varying matrix concentrations, electrophoresis, and staining/destaining of gels. In this paper a procedure is demonstrated which automatically generates all of the data required for a Ferguson plot using a replaceable sieving matrix (thereby eliminating gel polymerization) in a capillary format. In addition, this technique possesses the advantages inherent to capillary electrophoresis, namely, very fast separation times, and on-line monitoring which allows quantitation and precludes post-separation staining and destaining of gels.

Size-dependent separation of proteins denatured in SDS by capillary electrophoresis using a replaceable sieving matrix.

The determination of molecular weights by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most powerful electrophoretic techniques for protein characterization. A separation media has been developed which allows this type of analysis to be performed in the capillary format. A replaceable polymeric solution rather than a polymerized gel was used as the sieving matrix. This solution allowed for the separation of proteins denatured in SDS in a size-dependent manner as demonstrated by the linear correlation between the proteins' relative migration and the concentration of the sieving matrix (Ferguson plot). The logarithm of the molecular weight of protein standards correlated linearly with the relative mobility of the denatured proteins over the molecular weight range of 14,000 to 205,000. The calculated resolution at half-peak height was such that proteins that differed by as little as 4% in molecular weight would be resolved. Finally, the integrated peak areas at 215 nm were linearly proportional to the mass of the protein injected.

Analysis of the ligand-promoted global conformational change in aspartate transcarbamoylase. Evidence for a two-state transition from boundary spreading in sedimentation velocity experiments.

A global conformational change in the regulatory enzyme aspartate transcarbamoylase of Escherichia coli was demonstrated 20 years ago by the 3.5% decrease in the sedimentation coefficient of the enzyme upon its interaction with carbamoyl phosphate and saturating amounts of the aspartate analog succinate. This "swelling" of aspartate transcarbamoylase attributable to the T----R allosteric transition was observed also in subsequent studies when the enzyme was completely saturated with the bisubstrate analog N-(phosphonacetyl)-L-aspartate. In neither of these studies was a direct attempt made by an analysis of boundary spreading (expressed as an apparent diffusion coefficient) on partially liganded enzyme to determine whether the solution contained only T and R-state molecules, as expected for a concerted transition, or a mixture of more than two distinct conformational states. The sensitivity of boundary spreading measurements was tested with a known mixture of fully liganded wild-type enzyme (R-state) and an inactive T-state mutant that did not bind either succinate or the bisubstrate ligand. This experiment yielded broad boundaries with an apparent diffusion coefficient about 10% greater than that of T-state enzyme, due to the differential sedimentation of the two independent species. Identical boundary spreading was obtained theoretically by simulating an equimolar mixture of T and R-state aspartate transcarbamoylase. These results proved that the boundary spreading measurement was sensitive to the presence of heterogeneity. Analogous experiments with only wild-type enzyme in the presence of sub-stoichiometric amounts of the tightly bound bisubstrate ligand sufficient to promote a 1.8% decrease in sedimentation coefficient also exhibited broader boundaries, corresponding to a 10% increase in the apparent diffusion coefficient relative to the unliganded enzyme. In contrast, such broad boundaries were not observed in experiments when the weakly bound succinate was present in quantities sufficient to cause the same 1.8% decrease in sedimentation coefficient. The differences in boundary spreading observed with the two active-site ligands were accounted for by the affinities of the respective ligands for the enzyme and the transport theory of a ligand-promoted isomerization of the protein. In the presence of sub-stoichiometric levels of the tight-binding bisubstrate ligand, the dynamic equilibrium between the T and the R-state is essentially uncoupled and the species sediment at slightly different rates to give broad boundaries.(ABSTRACT TRUNCATED AT 250 WORDS)

Boundary spreading in sedimentation velocity experiments on partially liganded aspartate transcarbamoylase. A ligand-mediated isomerization.

Transport theory for rapidly reversible interacting systems was used to analyze boundary spreading in sedimentation velocity experiments on partially liganded aspartate transcarbamoylase. In the presence of sub-stoichiometric amounts of a bisubstrate analog, N-(phosphonacetyl)-L-aspartate, which is bound with high affinity to the enzyme (Kd approximately 100 nM), broad boundaries were observed consistent with the presence of two conformational forms. The theoretical treatment showed that under these conditions, the interconversion between the compact (11.7 S) and swollen (11.3 S) forms of the enzyme appears uncoupled, due to the formation of a gradient of free ligand that is caused by the re-equilibration resulting from the differential sedimentation of the two enzyme forms. Sedimentation velocity patterns for such systems are interpretable in terms of two independent species. When, however, the enzyme is in the presence of a sub-saturating amount of the weakly bound ligand, succinate (Kd approximately 1 mM), the re-equilibration caused by the differential sedimentation does not perturb the large background of free ligand and form a gradient. Instead, the two different forms of the enzyme are in dynamic equilibrium, resulting in a boundary having average sedimentation and diffusion coefficients. The observed boundary spreading experiments with different ligands are satisfactorily interpreted in terms of a ligand-mediated isomerization of aspartate transcarbamoylase from a compact to a swollen conformation.