Spectrophotometric Determination of Affinities of alpha-Factors for Their G Protein-Coupled Receptors in Saccharomyces cerevisiae

摘要

A new and relatively simple spectrophotometric technique has been developed for the accurate determination of alpha-factor pheromone affinities for Ste2p whole cell receptor in yeast a-cells. We designed and tested nine detector peptides containing mono- (epsilon(412)=14500) or tri-cysteine residues (epsilon(412)=43660). The free unbound detector was detected using Ellman's reagent at 412nm. Saturation binding studies using Saccharomyces cerevisiae Y 7925 (MATa) at a concentration of 2.5x10(11) cells/mL with the highest affinity detector 1, [Orn(6)]-factor-[Cys](3), resulted in a dissociation constant (K-D) of 1.67x10(-7) and total binding sites per cell (B-cell=29500 sites/cell) comparable with those obtained using radiolabeled binding assays. Competitive binding assay using five nonchromogenic -factor analogs allowed for the determination of each K-D value. [Orn(6),d-Ala(9)]-factor showed the highest receptor affinity (K-D=1.03x10(-7)M), which was threefold higher than that of native alpha-factor. This assay provides rapid and convenient results for determining the relative affinities of nonchromogenic alpha-factors and eliminates the need for radioactive waste disposal.