A new probe for affinity labelling pancreatic cholecystokinin receptor with minor modification of its structure

Eur J Biochem. 1989 Nov 6;185(2):397-403. doi: 10.1111/j.1432-1033.1989.tb15128.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels / chemical synthesis
  • Affinity Labels / metabolism*
  • Animals
  • Cholecystokinin / analogs & derivatives*
  • Cholecystokinin / chemical synthesis
  • Cholecystokinin / metabolism
  • Chromatography, Affinity
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Polyacrylamide Gel
  • Male
  • Pancreas / metabolism*
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / metabolism*
  • Photochemistry
  • Rats
  • Receptors, Cholecystokinin / metabolism*
  • Structure-Activity Relationship
  • Time Factors
  • Wheat Germ Agglutinins

Substances

  • Affinity Labels
  • Peptide Fragments
  • Receptors, Cholecystokinin
  • Wheat Germ Agglutinins
  • cholecystokinin (25-33),2-(4-azidosalicylamido)-1,3-dithiopropionate(Thr(28)-AHX(31))-
  • Cholecystokinin