Thirteen analogs of tridecapeptide α-factor (WHWLQLKPGQPMY) of Saccharomyces cerevisiae with C-or N-terminal Trp extension and isosteric replacement by Aib at position 8 and 11, Trp at position 13, D-Ala at position 9, and Orn and Glu at position 6 were synthesized and assayed for their biological activity. Receptor binding assay was carried out using our newly developed spectrophotometric method with detector peptide 14. C-or N-terminal extended analogs, α-factor-[Trp] n (n =1-5) 1-5 and [N-Trp] 1 -α-factor 6, were all less active than native α-factor and gradual decreases in both activity and receptor affinity were observed with greater Trp extension. Trp-substituted analog at position 13, [Trp 13 ]α-factor 7, exhibited about 2-fold reductions in both activity and receptor affinity. Aib-substituted analogs, [Aib 8 ]α-factor 8 and [Aib 11]α-factor 9, showed 5-to 10-fold reduction in activity as well as 3-fold reduction in receptor affinity compared to native α-factor. [Orn 6]α-factor 10 demonstrated strong potency with a 7.0-fold increase in halo activity as well as 1.8-fold increase in receptor affinity compared to native α-factor. ]α-factor 11 exhibited 15-fold higher halo activity as well as nearly 3-fold higher receptor affinity compared to native α-factor.

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‐ (ε412 = 14 500) or tri‐cysteine residues (ε412 = 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 × 1011 cells/mL with the highest affinity detector 1, [Orn6]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. [Orn6,d‐Ala9]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.

No abstract

No abstract

Ala 9]α-factor, and native α-factor (X = amino acids). Their biological activities (halo, gene induction, and affinity) were measured using S. cerevisiae Y7925 and LM102 and compared with those of native α-factor (100%). G protein-coupled receptor was expressed in strain LM102 containing pESC-LEU-STE2 vector. [Dap 6 ,D-Ala 9 ]α-factor with weak halo activity (10%) showed the highest receptor affinity (> 230%) and the highest gene induction activity (167%). [Arg 6 ,D-Ala 9 ]α-factor showed the highest halo activity (2,000%). The number of active binding sites per cell (about 20,000 for strain LM102) was determined using a newlydesigned fluorescence-based detector, [Arg 6 ,D-Ala 9 ]α-factor-Edan, with high sensitivity (12,500-fold higher than the absorption-based detector [Orn 6 ]α-factor-[Cys]3).