A B S T R A C T Experiments were designed to characterize several partial reactions of the Na,K-ATPase and to demonstrate that a model can be defined that reproduces most of the transport features of the pump with a single set of kinetic parameters. We used the fluorescence label 5-iodoacetamidofluorescein, which is thought to be sensitive to conformational changes, and the styryl dye RH 421, which can be applied to detect ion-binding and -release reactions. In addition transient electric currents were measured, which are associated mainly with the E l ~ E2 conformational transition. Numerical simulations were performed on the basis of a reaction model, that has been developed from the Post-Albers cycle. Analysis of the experimental data allows the determination of several rate constants of the pump cycle. Our conclusions may be summarized as follows: (a) binding of one Na + ion at the cytoplasmic face is electrogenic. This Na + ion is specifically bound to a neutral binding site with an affinity of 8 mM in the presence of 10 mM Mg 2+. In the absence of divalent cations, the intrinsic binding affinity was found to be 0.7 mM. (b) The analysis of fluorescence experiments with the cardiotonic steroid strophanthidin indicates that the 5-iodoacetamidofluorescein label monitors the conformational transition (Na3)EI-P ---, P-Ez(Na2), which is accompanied by the release of one Na + ion. 5-IAF does not respond to the release of the subsequent two Na + ions, which can be monitored by the RH 421 dye. These experiments indicate further that the conformational transition E,P ~ P-E2 is the rate limiting process of the Na + translocation. The corresponding rate constant was determined to be 22 s -1 at 20~ From competition experiments with cardiotonic steroids, we estimated that the remaining 2 Na § ions are released subsequently with a rate constant of at least 5,000 s -1 from their negatively charged binding sites. (c) Comparing the fluorescence experiments with electric current transients, which were performed at various Na concentrations in the absence and presence of strophanthidin, we found that the transition (Na3)'EI-P --, P-E2"(Na2) is the major charge translocating step in the reaction sequence Na3'El ~ (Na3)'E1-P ~ P-E2"(Na~) ~ P-E 2. The subsequent release of 2 Na + ions contributed less than 25% to the total electric current transient. 198 THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 104 9 1994 binding can be explained by a kinetic model. A quantitative description has been obtained under the assumption that these inhibitors bind only to the states P-E~(Na2) and P-E~ (K~). (e) Most of our experiments can be described by a modified Post-Albers scheme. A set of the kinetic parameters in this scheme has been determined by the experiments presented or by data from literature. Numerical simulations using this set are consistent with the presented data.

G protein-coupled receptors (GPCRs) constitute an abundant family of membrane receptors of high pharmacological interest. Cell-based assays are the predominant means of assessing GPCR activation, but are limited by their inherent complexity. Functional molecular assays that directly and specifically report G protein activation by receptors could offer substantial advantages. We present an approach to immobilize receptors stably and with defined orientation to substrates. By surface plasmon resonance (SPR), we were able to follow ligand binding, G protein activation, and receptor deactivation of a representative GPCR, bovine rhodopsin. Microcontact printing was used to produce micrometer-sized patterns with high contrast in receptor activity. These patterns can be used for local referencing to enhance the sensitivity of chip-based assays. The immobilized receptor was stable both for hours and during several activation cycles. A ligand dose-response curve with the photoactivatable agonist 11-cis-retinal showed a half-maximal signal at 120 nM. Our findings may be useful to develop novel assay formats for GPCRs based on receptor immobilization to solid supports, particularly to sensor surfaces.

Rhodopsin-transducin coupling was used as an assay to investigate a laterally patterned membrane reconstituted with a receptor and its G protein. It served as a model system to show the feasibility to immobilize G protein-coupled receptors on solid supports and investigate receptor activation and interaction with G proteins by one-dimensional imaging surface plasmon resonance. Supported membranes were formed by the self-assembly of lipids and rhodopsin from detergent solution onto functionalized gold surfaces. They formed micrometer-sized alternating regions of pure fluid phospholipid bilayers separated by bilayers composed of an outer phospholipid leaflet on a gold-attached inner thiolipid. Rhodopsin was found to incorporate preferentially into the phospholipid bilayer regions, whereas transducin was uniformly distributed over the entire outer surface of the supported patterned membrane. The influence of rhodopsin on the dark binding of transducin to lipid membranes was described quantitatively and compared with previously published data. Coupling reactions with transducin resembled closely the native system, indicating that the native functionality of rhodopsin was preserved in the supported membranes. The spatially varying properties of the membranes resulted in a pattern of rhodopsin activity on the surface. This combination of techniques is very promising for the investigation of the lateral diffusion of transducin, can be extended to include signalling proteins downstream of the G protein, and may be applied to functional screening of other G protein-coupled receptors. In the future, it may also serve as a basis for constructing biosensors based on receptor proteins.

No abstract

A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass-like surface.In a first step, phospholipids carrying head-group thiols were covalently immobilized onto Si02-Ti02 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thioethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50-60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 A , which corresponds to the thickness of a fluid bilayer membrane.Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipidanchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopeptide concentration in the outer monolayer.

). Thus, this very sensitive assay provides the first evidence that the function of SC in sIgA complex is not to modify the affinity for the antigen. K A falls to 6.6 ؋ 10 5 M ؊1 when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is critical for recognition by the antibody.

Recent technological advances in high-content screening instrumentation have increased its ease of use and throughput, expanding the application of high-content screening to the early stages of drug discovery. However, high-content screens produce complex data sets, presenting a challenge for both extraction and interpretation of meaningful information. This shifts the high-content screening process bottleneck from the experimental to the analytical stage. In this article, the authors discuss different approaches of data analysis, using a phenotypic neurite outgrowth screen as an example. Distance measurements and hierarchical clustering methods lead to a profound understanding of different high-content screening readouts. In addition, the authors introduce a hit selection procedure based on machine learning methods and demonstrate that this method increases the hit verification rate significantly (up to a factor of 5), compared to conventional hit selection based on single readouts only. (Journal of Biomolecular

High-Throughput Screening (HTS) data in its entirety is valuable raw material for the drug-discovery process. It provides the most complete information about the biological activity of a company's compounds. However, its quantity, complexity and heterogeneity require novel, sophisticated approaches in data analysis. At GeneData, we are developing methods for large-scale, synoptical mining of screening data in a five-step analysis:(1) Quality Assurance: Checking data for experimental artifacts and eliminating low quality data.(2) Biological Profiling: Clustering and ranking of compounds based on their biological activity, taking into account specific characteristics of HTS data.(3) Rule-based Classification: Applying user-defined rules to biological and chemical properties, and providing hypotheses on the biological mode-of-action of compounds.(4) Joint Biological-Chemical Analysis: Associating chemical compound data to HTS data, providing hypotheses for structure-activity relationships.(5) Integration with Genomic and Gene Expression Data: Linking into other components of GeneData's bioinformatics platform, and assessing the compounds' modes-of-action, toxicity, and metabolic properties. These analyses address issues that are crucial for a correct interpretation and full exploitation of screening data. They lead to a sound rating of assays and compounds at an early stage of the lead-finding process.