vasoactive-intestinal-peptide and 4-azidophenylglyoxal

Molecular characterization of receptors depends on the availability of ligand derivatives carrying a reactive group to covalently link the active sites. Two vasoactive intestinal peptide (VIP) derivatives, each labeled either at the two arginine residues 12 and 14 or singly in position 14, were prepared. In the first case, this was achieved by a selective chemical modification using azidophenylglyoxal. In the second, the amino acids of VIP, buried in the active site of the receptor, were protected and one arginine residue of bound VIP was successfully modified using azidophenylglyoxal. The two molecules were resolved by radioimmunocompetition and reversed phase high performance liquid chromatography. Identification of sites of labeling was achieved by tryptic peptide mapping and amino acid analysis. One derivative (Az-Bz-Arg14-VIP) retains a high binding affinity for the receptor and was found to be biologically active. The present method yields a derivative which is useful in structural analysis of the receptor.

Topics: Aldehydes; Amino Acids; Animals; Arginine; Azides; Binding, Competitive; Cross-Linking Reagents; Cyclic AMP; Guanidine; Guanidines; In Vitro Techniques; Phenylglyoxal; Photochemistry; Rats; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

The biological effects of vasoactive intestinal peptide (VIP) are mediated by binding to a membrane-bound receptor. Probes designed to trap this receptor by binding to it in a covalent way may suffer from a greatly reduced affinity. We report here, for the VIP receptor, the use of a photoaffinity probe obtained by derivatization of receptor-bound VIP with para-azidophenylglyoxal. This method protected the parts of the molecule essential for receptor binding. The VIP derivative thus obtained became covalently linked when irradiated. In the dark, however, it exhibited normal VIP-like behavior and retained its biological activity. This derivatization method might be generally applicable when hormone analogues have to be prepared without loss of receptor affinity. Receptor characterization studies on liver plasma membranes showed the presence of high- and low-affinity binding sites with KD = 0.1 and 5 nM, respectively. Treatment of membranes with dithiothreitol causes loss of high-affinity binding. The high-affinity site, trapped by the photoaffinity probe, resolved into two molecular mass forms, 50 and 200-250 kDa. Reduction of the receptor-probe complex left the 50-kDa form intact, whereas the amount of the 200-250-kDa form greatly diminished. We demonstrate the importance of the presence of disulfide bonds in one of the molecular forms involved in high-affinity binding.

Topics: Affinity Labels; Amino Acids; Animals; Azides; Chromatography, High Pressure Liquid; Kinetics; Male; Molecular Weight; Phenylglyoxal; Photochemistry; Rats; Rats, Inbred Strains; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Substrate Specificity; Vasoactive Intestinal Peptide