Photoaffinity labeling of the cerebral sulfonylurea receptor using a novel radioiodinated azidoglibenclamide analogue.
In previous studies evidence has been presented by photoaffinity labeling that a polypeptide of 145-150 kDa represents the cerebral sulfonylurea receptor. However, covalent incorporation of [3H]glibenclamide or a 125I-labeled glibenclamide analogue into the sulfonylurea receptor required high amounts of photoenergy and took place with low yield of photoinsertion. To provide a probe with increased photoreactivity a 4-azido-5-iodosalicyloyl analogue of glibenclamide was synthesized. Binding experiments revealed specific and reversible high-affinity binding of this novel probe to the particulate (KD = 0.13 nM) and solubilized (KD = 0.56 nM) sulfonylurea receptor from cerebral cortex. The novel probe showed > 100-fold higher sensitivity to irradiation at 356 nm than glibenclamide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific photoincorporation into a cerebral protein of 175 kDa and indicated an efficiency of photoincorporation of 9%. From dissociation binding curves following irradiation photoincorporation was estimated as 28% of specifically bound ligand. Photoincorporation into the 175-kDa protein following saturation binding of the novel probe to particulate sites from cerebral cortex indicated a KD value of 0.38 nM. Inhibition of photoincorporation into this protein by glibenclamide, glipizide, and tolbutamide revealed KD values for these sulfonylureas of 0.06 nM, 1.6 nM, and 1.2 microM, respectively. These results show that the novel photoaffinity ligand can be used as a probe for detection and characterization of the sulfonylurea receptor and suggest that a 175-kDa protein represents the cerebral sulfonylurea receptor.
Institute of Pharmacology and Toxicology, University of Göttingen, Germany.
M
M
Schwanstecher
S
S
Löser
F
F
Chudziak
C
C
Bachmann
U
U
Panten
D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002458:Cell Fractionation; D002540:Cerebral Cortex / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005913:Glipizide / Q000494:pharmacology; D005905:Glyburide / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D008970:Molecular Weight; D010782:Photolysis; D015221:Potassium Channels / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011869:Radioligand Assay; D011955:Receptors, Drug / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D064233:Sulfonylurea Receptors; D013552:Swine; D014044:Tolbutamide / Q000494:pharmacology; D014316:Tritium
C088346:4-azido-5-iodosalicyloylglibenclamide; D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D007457:Iodine Radioisotopes; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D014316:Tritium; D014044:Tolbutamide; D005905:Glyburide; D005913:Glipizide