A new probe for affinity labelling pancreatic cholecystokinin receptor with minor modification of its structure.
Biochemical studies on receptors for peptides are most often carried out on affinity-labelled (peptide-receptor) complexes. Necessarily, the assumption is made that a covalent (peptide-receptor) complex behaves as the native receptor. The validity of this assumption is dependent on both the affinity-labelling technique and the resolution of the analytical method used for biochemical characterization. We designed a new affinity-labelling probe in order to minimize structural modifications occurring within the affinity-labelled cholecystokinin (CCK) receptor protein. The probe was 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK-25-33, (125I-ASD-[Thr28,Ahx31]CCK-25-33), the peptide moiety of which was released from its binding site by reduction. It was obtained by coupling a photoactivable chemical to [Thr28,Ahx31]CCK-25-33 via its N-terminus. The resulting peptide was HPLC purified and radioiodinated in the presence of chloramine T. Binding of 125I-ASD-[Thr28,Ahx31]CCK-25-33 was time- and temperature-dependent and reversible. At 25 degrees C, a steady-state level was reached after 60 min and half-maximal dissociation after 38 min. Binding was inhibited by [Thr28,Ahx31]CCK-25-33 and L-364-718 antagonist with IC50 0.4 nM and 0.9 nM, respectively. Photoaffinity labelling of pancreatic plasma membranes by 125I-ASD-[Thr28,Ahx31]CCK-25-33 identified a glycoprotein of Mr 85,000-100,000 which was retained on immobilized wheat germ agglutinin. Enzyme cleavage by endoproteinase Glu-C generated a main fragment of Mr 30,000-34,000. The same glycoprotein was photoaffinity labelled with 125I-DTyr-Gly-[Ahx28,31,pNO2Phe33]CCK-26-33 (Ahx, 2-aminohexanoic acid; pNO2Phe,p-nitrophenylalanine) an intrinsic probe having its photolabile group sited in the binding domain of cholecystokinin. 125I-ASD-[Thr28,Ahx31]CCK-25-33 is a potentially powerful tool for biologically and biochemically studying cholecystokinin receptors.
Fourmy
D
D
Groupe de Recherche de Biologie et Pathologie Digestine, INSERM U 151, CHU Rangueil, Toulouse, France.
Lopez
P
P
Poirot
S
S
Jimenez
J
J
Dufresne
M
M
Moroder
L
L
Powers
S P
SP
Vaysse
N
N
D000345:Affinity Labels / Q000138:chemical synthesis / Q000378:metabolism*; D000818:Animals; D002766:Cholecystokinin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D010179:Pancreas / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D010777:Photochemistry; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013997:Time Factors; D014909:Wheat Germ Agglutinins
D000345:Affinity Labels; D010446:Peptide Fragments; D011949:Receptors, Cholecystokinin; D014909:Wheat Germ Agglutinins; C061851:cholecystokinin (25-33),2-(4-azidosalicylamido)-1,3-dithiopropionate(Thr(28)-AHX(31))-; D002766:Cholecystokinin