Angiopoietin-1 promotes functional neovascularization that relieves ischemia by improving regional reperfusion in a swine chronic myocardial ischemia model

10.1007/s11373-006-9082-xJournal of Biomedical Science134579-59

A radioimmunoassay to screen for antibodies to native conformational antigens and analyse ligand-induced structural states of antigenic proteins

A radioimmunoassay is described in which antigenic protein was immobilized by incubating nitrocellulose filters of defined diameter with antigen-containing solutions. The amount of adsorbed antigen increased in a linear fashion over a wide range of antigen concentrations. The antigen-antibody reactions and the indicator reactions were performed by incubating the filters with appropriate solutions. During the test any contact of the antigen with air was avoided. Antigenic sites which are sensitive to protein denaturation by drying could be detected with the assay. The assay was also used to screen hybridoma supernatants for antibodies directed against Na+ cotransport proteins from renal brush-border membranes. Monoclonal antibodies were selected which showed different binding characteristics depending on whether or not substrates of Na+ cotransporters were present. Since binding of several antibodies was altered by two different substrates and not by non-transported control substances, these monoclonal antibodies were believed to interact with more than one transport system. One of the antibodies, which showed different antibody binding after addition of D-glucose or L-lactate, bound to a polypeptide component of the renal Na+-D-glucose cotransporter and was able to inhibit Na+ gradient-dependent D-glucose uptake in brush-border membrane vesicles (Koepsell, Korn, Raszeja-Specht, Bernotat-Danielowski, Ollig, 1988, J. Biol. Chem., in press). To investigate the effects of D-glucose and L-lactate on the binding of this antibody concentration dependence was measured. High and low affinity binding sites for D-glucose and L-lactate were characterized thereby demonstrating that the radioimmunoassay permits investigations of the properties of high and low affinity substrate binding site

Monoclonal antibodies against the renal Na⁺-D-glucose cotransporter. Identification of antigenic polypeptides and demonstration of functional coupling of different Na⁺-cotransport systems

Eight monoclonal antibodies are described which are directed against the renal Na+-D-glucose cotransporter. In porcine renal brush-border membranes, the antibodies either bind to one or to three polypeptides which have been identified as components of the Na+-D-glucose cotransporter (Neeb, M., Kunz, U., and Koepsell, H., (1987) J. Biol. Chem. 262, 10718-10727). Their molecular weights and isoelectric points are 75,000 and pH 5.5, 60,000 and pH 5.2, and 47,000 and pH 5.4. Six antibodies were able to influence Na+-dependent D-glucose uptake and/or Na+-dependent high affinity phlorizin binding. In the presence of Na+, the binding of all antibodies to native membrane proteins was altered by D-glucose but not by D-mannose. Since this effect was observed with D-glucose concentrations less than 1 x 10(-8) M, a high affinity D-glucose-binding site on the D-glucose transporter has been implied. Some of the antibodies probably interact also with other Na+-coupled transporters since their binding was altered by micromolar concentrations of L-lactate, L-alanine, or L-glutamate but not by the nontransported control substances D-alanine and D-glutamate. L-lactate increased the binding of one antibody in the absence but not in the presence of D-glucose. Effects of L-lactate and L-alanine on the binding of another antibody were only observed when D-glucose was present. Thus, some epitopes on the Na+-D-glucose cotransporter are altered by D-glucose and also by substrates of other Na+ cotransporters. This finding suggests functional coupling of different Na+-cotransport systems