Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic cholecystokinin receptor

• Published in: Biochemistry (Easton) 1989-04, Vol.28 (8), p.3463-3468
• Main Authors: Klueppelberg, Ulrich G, Gaisano, Herbert Y, Powers, Stephen P, Miller, Laurence J
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; Affinity Labels; ALKALI METAL COMPOUNDS; AMINO ACIDS; Animals; AROMATICS; AZAARENES; AZOLES; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; Binding Sites; Binding, Competitive; BODY; CARBOXYLIC ACIDS; CELL CONSTITUENTS; CELL MEMBRANES; CHEMICAL REACTIONS; Cholecystokinin - metabolism; CHROMATOGRAPHY; CROSS-LINKING; DAYS LIVING RADIOISOTOPES; DIGESTIVE SYSTEM; ELECTRON CAPTURE RADIOISOTOPES; ENDOCRINE GLANDS; GLANDS; HALIDES; HALOGEN COMPOUNDS; HALOGENATION; HETEROCYCLIC ACIDS; HETEROCYCLIC COMPOUNDS; In Vitro Techniques; INDOLES; INORGANIC PHOSPHORS; INTERMEDIATE MASS NUCLEI; IODIDES; IODINATION; IODINE 125; IODINE COMPOUNDS; IODINE ISOTOPES; ISOTOPES; KININS; LABELLING; LIGANDS; LIQUID COLUMN CHROMATOGRAPHY; Male; MEMBRANE PROTEINS; Membrane Proteins - metabolism; MEMBRANES; MOLECULAR STRUCTURE; Molecular Weight; NUCLEI; ODD-EVEN NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; PANCREAS; Pancreas - metabolism; PEPTIDES; PHOSPHORS; POLYMERIZATION; POLYPEPTIDES; PROTEINS; PYRROLES; RADIOISOTOPES; Rats; RECEPTORS; Receptors, Cholecystokinin - metabolism; SEPARATION PROCESSES; SODIUM COMPOUNDS; SODIUM IODIDES; TRYPTOPHAN; Tryptophan - analogs & derivatives
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00434a047

We report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an Mr = 85,000-95,000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same Mr = 85,000-95,000 pancreatic membrane protein. This probe, 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp30)CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity (Kd = 3 nM). While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion (EC50 = 1 nM) and equally efficacious to native hormone. Despite the slight decrease in affinity, this probe demonstrated a high relative efficiency of covalent labeling of the Mr = 85,000-95,000 protein. This confirms that the Mr = 85,000-95,000 protein represents the hormone-binding subunit of the CCK receptor and demonstrates the utility of this type of photoaffinity labeling probe.