Identification of ligand binding regions of the Saccharomyces cerevisiae alpha-factor pheromone receptor by photoaffinity cross-linking

Date

2004-10-19

Author

Son, Çağdaş Devrim
Naider, F
Becker, JM

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

41
views

0
downloads

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), a photoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on a p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of alpha-factor to generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growth arrest assay) and binding affinities of all analogues for the alpha-factor receptor (Ste2p) were determined. Two of the analogues that were tested, Bpa(1) and Bpa(5), showed 3-4-fold lower affinity than the alpha-factor, whereas Bpa(3) and Bpa(13) had 7-12-fold lower affinities. Bpa(8) competed poorly with [H-3]-a-factor for Ste2p. All of the analogues tested except Bpa(8) had detectable halos in the growth arrest assay, indicating that these analogues are alpha-factor agonists. Cross-linking studies demonstrated that [Bpa(1)]-alpha-factor, [Bpa(3)]-alpha-factor, [Bpa(5)]-alpha-factor, and [Bpa(13)]-alpha-factor were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa(1), Bpa(3), and Bpa(13) cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of the pheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between the position 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The results further define the sites of interaction between Ste2p and the alpha-factor, allowing refinement of a model for the pheromone bound to its receptor.

Subject Keywords

Biochemistry

URI

https://hdl.handle.net/11511/42592

Journal

BIOCHEMISTRY

DOI

https://doi.org/10.1021/bi0496889

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

Determination of trace elements in marine plankton by inductively coupled plasma mass spectrometry (ICP-MS) Arslan, Z; Ertas, N; Tyson, JF; Uden, PC; Denoyer, ER (Springer Science and Business Media LLC, 2000-02-01) A method has been developed for the determination of 23 elements in marine plankton in which inductively coupled plasma (ICP) source mass spectrometry (MS) was used to quantify the elements in the solution after digestion in a mixture of hydrofluoric and nitric acids in sealed PTFE vessels in a microwave field. The procedure was validated by the analysis of a standard reference soil (SRM 2709 San Joaquin Soil) and a standard reference fresh water plankton (CRM 414). The method was applied to the analysis of...
Isolation and immunological characterization of theta class glutathione-s-transferase gstt2-2 from bovine liver İşgör, Sultan Belgin; Çoruh, Nursen; Department of Biochemistry (2004) The glutathione-S-transferases (GSTs) (EC.2.5.1.18) are enzymes that participate in cellular detoxification of endogenous as well as foreign electrophilic compounds, function in the cellular detoxification systems and are evolved to protect cells against reactive oxygen metabolites by conjugating the reactive molecules to the nucleophile scavenging tripeptide glutathione (GSH, ?-glu-cys-gly). The GSTs are found in all eukaryotes and prokaryotic systems, in the cytoplasm, on the microsomes, and in the mitoch...
Investigation for natural extract inhibitors of bovine lens aldose reductase responsible for the formation of diabetis dependent cataract Onay, Melih; Çoruh, Nursen; Department of Biochemistry (2008) In the polyol pathway, Aldose reductase (AR) is an important enzyme in reduction of aldehydes and aldosugars to their suitable alcohols. AR, using NADPH as a coenzyme, has a molecular weight of 37 000 dalton. AR in its activated form, known to increase the sorbitol accumulation in lens, is responsible for the cataract formation in diabetis diseases. Therefore, the inhibition of aldose reductase is important to prevent the incedence of cataract formation in diabetus mellitus. In the treatment of diabetis dep...
Purification of glutathione S-transferases and genetic characterization of Zeta isozyme from Pinus brutia, Ten Öztetik, Elif; İşcan, Mesude; Department of Biochemistry (2005) Glutathione S-transferases (GST, EC2.5.1.18) are a family of multifunctional, dimeric enzymes that catalyse the nucleophilic attack of the tripeptide glutathione (?-L-glutamyl-L-cysteinyl-L-glycine) on lipophilic compounds with electrophilic centres. The primary function of GSTs is generally considered to be the detoxification of both endogenous and xenobiotic compounds. Cytosolic GSTs have been grouped into eleven distinct classes as: (A); Alpha, (M); Mu, (P); Pi, (S); Sigma, (T); Theta, (Z); Zeta, (F); Ph...
POSSIBLE INVOLVEMENT OF MANGANESE IN THE CATALYTIC MECHANISM OF BOVINE LIVER ARGINASE TURKOGLU, S; OZER, I (Elsevier BV, 1992-06-01) 1. Bovine liver arginase followed Michaelis-Menten kinetics in the pH range of 4.5-9.0. The variation of upsilon(i) pH implied that a basic group (pK(alpha) 8.7) functions at the catalytic site.

Citation Formats

Ç. D. Son, F. Naider, and J. Becker, “Identification of ligand binding regions of the Saccharomyces cerevisiae alpha-factor pheromone receptor by photoaffinity cross-linking,” BIOCHEMISTRY, pp. 13193–13203, 2004, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/42592.