Photoaffinity labeling of the glucagon receptor with a new glucagon analog.
The preparation, purification and characterization of N epsilon-4- azidophenylamidinoglucagon are described. This photoreactive peptide was found to be 50% as potent as native glucagon in competing with 125I-labeled glucagon for binding to glucagon receptors on rat liver plasma membranes. Similarly, the analog was 50% as potent as native glucagon in its ability to stimulate adenylate cyclase. The photoreactive glucagon analog was radioiodinated to high specific activity with iodine-125 and was used to label rat liver plasma membrane proteins. Analysis of labeled membrane proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed covalent incorporation predominantly into a protein of relative molecular mass, Mr, of 50 000-60 000. Occasionally a protein of Mr 170 000-180 000 was also labeled. Irradiation of membranes in the presence of unlabeled glucagon or GTP selectively inhibited the labeling of the 50 000-60 000-Mr protein(s). As a result of these studies we suggest that the sodium-dodecyl-sulfate-dissociated glucagon receptor is a 50 000-60 000-Mr protein.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000345:Affinity Labels / Q000138:chemical synthesis* / Q000302:isolation & purification; D000818:Animals; D001345:Autoradiography; D001386:Azides / Q000138:chemical synthesis* / Q000302:isolation & purification; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005934:Glucagon / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D010777:Photochemistry; D051381:Rats; D011956:Receptors, Cell Surface / Q000032:analysis*; D018027:Receptors, Glucagon
D000345:Affinity Labels; D001386:Azides; D011956:Receptors, Cell Surface; D018027:Receptors, Glucagon; C041462:glucagon, 12-(N(6)-(4-azidophenylamidino)Lys)-; D005934:Glucagon; D000262:Adenylyl Cyclases