Protease peptide mapping of affinity-labeled rat pancreatic cholecystokinin-binding proteins.
Affinity-labeling probes with sites of cross-linking distributed along the ligand have been used to biochemically characterize the pancreatic cholecystokinin (CCK) receptor. Probes with photolabile sites spanning the receptor-binding domain have labeled a Mr = 85,000-95,000 plasma membrane protein, while a probe cross-linked via the amino terminus of CCK-33, far removed from the carboxyl-terminal receptor-binding domain, has labeled a distinct Mr = 80,000 protein. In this work, protease peptide mapping of the pancreatic proteins labeled by each of these probes has been performed to gain insight into the identities of the bands and to define domains of the labeled proteins. Photolabile decapeptide probes with sites of cross-linking at the amino terminus, mid region, and carboxyl terminus of the receptor-binding domain each labeled a Mr = 85,000-95,000 glycoprotein with a Mr = 42,000 core protein and similar Staphylococcus aureus V8 protease peptide maps. This confirms that each probe labels the same binding protein and the same domain of that protein. Serial slices through the broad labeled band were separately deglycosylated and protease-treated, demonstrating a single protein core with differential glycosylation. The CCK-33-based probe, however, labeled predominantly two proteins, one having similar sizes in its native and deglycosylated forms to that labeled by the decapeptide probes and a distinct Mr = 80,000 protein. Of note, the peptide map of the protein believed to be the same as that labeled by the shorter probes was different, suggesting that this probe labeled the binding subunit at a site distinct from that which was labeled by the short probes.
Klueppelberg
U G
UG
Gastroenterology Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
Powers
S P
SP
Miller
L J
LJ
D000345:Affinity Labels / Q000138:chemical synthesis / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D051381:Rats; D011919:Rats, Inbred Strains; D011949:Receptors, Cholecystokinin / Q000302:isolation & purification* / Q000378:metabolism; D012697:Serine Endopeptidases
D000345:Affinity Labels; D010446:Peptide Fragments; D011949:Receptors, Cholecystokinin; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase