In Vitro Binding Assay of ³¹Methionine-oxidized Cholecystokinin Octapeptide to the CCK_B Receptor

Abstract

Methionine (Met) residues of cholecystokinin octapeptide (CCK8) are easily oxidized to produce Met sulfoxide and/or sulfone of CCK8. In order to investigate the effect of modification at Met of CCK8 for its CCK_B receptor, we evaluated the binding affinity of 4 oxidized forms of CCK8 for CCK_B receptor expressed in the plasma membrane of LoVo cells, by performing a flow cytometry-based competitive binding assay using fluorescein isothiocyanate (FITC)-labeled CCK8. Oxidative modification of CCK8 at ²⁸Met and ³¹Met caused to increase its binding affinity. The affinity of ³¹Met sulfone CCK8 was strongest and was significantly higher (by 20-30%) than that of native CCK8 (p<0.05). In contrast, the binding affinity of modified CCK8 was attenuated on replacing ³¹Met with ³¹Leu, ³¹Lys, or ³¹His.