Photoreactive insulin derivatives for the detection of the doubly labeled insulin receptor.
Two different photoinsulins, the radioactive N(epsilonB29)-(4-azidosalicyloyl) insulin and the novel biotinylated N(epsilonB29)-(4-azidotetrafluorobenzoyl-biocytinyl) insulin, were synthesized in order to study the binding stoichiometry of insulin and the insulin receptor in a direct approach. Both derivatives were cross-linked simultaneously to the (alphabeta)(2) receptor. Insulin-receptor conjugates were formed that carry a radioactive label as well as a biotin label. The doubly labeled complexes were isolated by streptavidin-affinity chromatography. Analysis of both markers, the radioactive (125)I marker and the biotin marker, proved the existence of a covalent complex of one receptor molecule and two ligands. Thus, orthogonal photocross-linking is introduced as a method for the isolation and analysis of bivalent receptor complexes.
Kleinjung
J
J
German Wool Research Institute, University of Technology Aachen, Veltmanplatz 8, 52062, Aachen, Germany.
Fabry
M
M
D001386:Azides / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D019921:Biotinylation; D002460:Cell Line; D003432:Cross-Linking Reagents; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000493:pharmacokinetics; D007457:Iodine Radioisotopes; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism; D046911:Macromolecular Substances; D010777:Photochemistry; D011869:Radioligand Assay; D011972:Receptor, Insulin / Q000032:analysis* / Q000378:metabolism
D001386:Azides; D003432:Cross-Linking Reagents; D007328:Insulin; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; C412642:N(epsilon)B29-(4-azidosalicyloyl) insulin; C412643:N(epsilon)B29-(4-azidotetrafluorobenzoyl-biocytinyl) insulin; D011972:Receptor, Insulin