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Spectrophotometric Determination of Affinities of α‐Factors for Their G Protein‐Coupled Receptors in Saccharomyces cerevisiae
Author(s) -
Ahn Hee Jun,
Kim Hyung Jin,
Jin Dong Hoon,
Hong Nam Joo
Publication year - 2015
Publication title -
bulletin of the korean chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.237
H-Index - 59
ISSN - 1229-5949
DOI - 10.1002/bkcs.10367
Subject(s) - affinities , saccharomyces cerevisiae , chemistry , dissociation constant , receptor , yeast , competitive binding , cysteine , binding affinities , chromatography , stereochemistry , biochemistry , biophysics , biology , enzyme
A new and relatively simple spectrophotometric technique has been developed for the accurate determination of α‐factor pheromone affinities for Ste2p whole cell receptor in yeast a ‐cells. We designed and tested nine detector peptides containing mono‐ (ε 412 = 14 500) or tri‐cysteine residues (ε 412 = 43 660). The free unbound detector was detected using Ellman's reagent at 412 nm. Saturation binding studies using Saccharomyces cerevisiae Y 7925 ( MATa ) at a concentration of 2.5 × 10 11 cells/ mL with the highest affinity detector 1 , [Orn 6 ]α‐factor‐[Cys] 3 , resulted in a dissociation constant ( K D ) of 1.67 × 10 −7 and total binding sites per cell ( B cell = 29 500 sites/cell) comparable with those obtained using radiolabeled binding assays. Competitive binding assay using five nonchromogenic α‐factor analogs allowed for the determination of each K D value. [Orn 6 , d ‐Ala 9 ]α‐factor showed the highest receptor affinity ( K D = 1.03 × 10 −7 M), which was threefold higher than that of native α‐factor. This assay provides rapid and convenient results for determining the relative affinities of nonchromogenic α‐factors and eliminates the need for radioactive waste disposal.