Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic cholecystokinin receptor.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Klueppelberg
U G
UG
Gastroenterology Research Unit, Mayo Clinic, Rochester, Minnesota 55905.
Gaisano
H Y
HY
Powers
S P
SP
Miller
L J
LJ
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002766:Cholecystokinin / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism*; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives
D000345:Affinity Labels; D008565:Membrane Proteins; D011949:Receptors, Cholecystokinin; C055424:6-nitrotryptophan; D014364:Tryptophan; D002766:Cholecystokinin