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PubMed2002

Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling.

Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiaewas used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of alpha-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following characterization, one analogue, [Bpa(1), Tyr(3), Arg(7), Phe(13)]alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor. Binding of the di-iodinated probe was saturable (K(d) = 200 nM) and competable by alpha-factor. Cross-linking into Ste2p was specific for this receptor and reversed by the wild-type pheromone. Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7. This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor. After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe. This is the first determination of a specific contact region between a Class IV GPCR and its ligand. The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR.

Affinity Labels↗
PubMed1996

Photoaffinity labeling of the active site of the Na+/K(+)-ATPase with 4-azido-2-nitrophenyl phosphate.

Na+/K(+)-ATPase will hydrolyze small acylphosphates such as p-nitrophenyl phosphate (pNPP) in addition to ATP and can derive sufficient energy from the hydrolysis of these small molecules to catalyze active ion transport. In this report, 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive analog of pNPP, was used as a probe of the substrate binding site of dog renal Na+/K(+)-ATPase. ANPP was slowly hydrolyzed by Na+/K(+)-ATPase with a Vmax of 0.19 mumol mg-1 min-1 and with an apparent Km of 1.0 mM. The Km for hydrolysis of pNPP was 1.7 mM. ANPP competitively inhibited the hydrolysis of pNPP with a Ki of 0.37 mM. Both the ATPase and pNPPase activity of the Na+/K(+)-ATPase were irreversibly inhibited after photolysis of the enzyme and ANPP with UV light, although neither activity was completely inhibited by up to 200 microM ANPP. Inhibition of activity was prevented by including 0.2 mM ATP in the reaction or by excluding Mg2+ from the photolysis buffer. Photolysis with [32P]ANPP labeled only the alpha subunit of the Na+/K(+)-ATPase, and the amount of labeling was substantially reduced by 0.2 mM ATP or in the absence of Mg2+. The stoichiometry of labeling extrapolated to a maximum of about 1.2 nmol/mg of protein at 100% inhibition of Mg(2+)-dependent activity. Limited proteolytic digestion showed labeling sites on nonoverlapping tryptic peptides derived from the alpha subunit of Na+/K(+)-ATPase, and two radiolabeled peptides were purified from an exhaustive tryptic digest of [32P]ANPP-labeled Na+/K(+)-ATPase. One peptide contained amino acids Met-379 to Lys-406, and the second contained amino acids Ala-655 to Lys-676. Amino acids corresponding to Asn-398 and Pro-668 were missing from the sequences and may represent residues derivatized by ANPP from within the substrate binding site of Na+/K(+)-ATPase.

Affinity Labels↗
PubMed1979

Photoaffinity labeling of the angiotensin II receptor. 3. Receptor inactivation with photolabile hormone analogues.

It has been shown that the receptor of angiotensin II (AT) in rabbit aorta strips, rat aorta, and rat stomach can be blocked specifically and irreversibly by several photolabile analogues of Sar-Arg-Val-Tyr-Val-His-Pro-Phe ([Sar1]AT) with irradiation. The effectiveness of a photolabel with light of wavelength 365 nm depends on the labeling amino acid (L-4'-nitrophenylalanine, L-4'-diazoniumphenylalanine, or L-4'-azidophenylalanine) and on its position in the peptide (replacing Tyr4 and/or Phe8). The 4'-azido)Phe-containing analogues are all good to fair photoinactivators. Their decreasing order of effectiveness is as follows: [Sar1,(4'-azido)Phe8]AT, [Sar1,(4'-azido)Phe4,8]AT, and [Sar1,(4'-azidoPhe4]AT. The (4'-nitro)Phe analogues show the opposite relation: the good ligand [Sar1,(4'-nitro)Phe8]AT is almost ineffective, but the nonligand [Sar1,(4-nitro)Phe4]AT exhibits good, specific photoinactivation. This can be explained by the existence of a different photolysis pathway for (4'-nitro)Phe: this analogue probably undergoes a multiphoton decay with a long-lived first excited state. A peptide in this state may differ in its pharmacological properties from the ground state and become a ligand.

Affinity Labels↗
PubMed1982

9-azidoacridine, a new photoaffinity label for nucleotide- and aromatic-binding sites in proteins.

The effect of the photolytic reagent 9-azidoacridine, optionally 3H-labelled, was studied both kinetically and structurally on nine different enzymes, namely alpha-chymotrypsin, lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, alcohol dehydrogenase, alanine dehydrogenase, D-amino acid oxidase, ribonuclease A, alkaline phosphatase and alpha-amylase. Dark inhibition was observed in several cases. The concentration of the inhibitor ranged from 0.2 microM to 0.67 microM and demonstrated competitive kinetics with nucleotide cofactors when present. All concentrations of inhibitor showed increased inhibition on photolysis. Examination of the oligopeptides from hydrolysis of the covalently 3H-labelled derivative in conjunction with known amino acid sequence and tertiary structure established that the primary site of interaction in those cases for which the tertiary structure was available involved the active-site region. The above results in conjunction with those obtained with the structural analogues 9-aminoacridine and 9-amino-1,2,3,4-tetrahydroacridine established that this reagent acts as a molecular probe of aromatic- and, in particular, nucleotide-binding sites. This reagent provides a further additional method for studying the nucleotide cofactor domain.

Affinity Labels↗
PubMed1984

Photoaffinity labelling of nucleoside-transport proteins in plasma membranes isolated from rat and guinea-pig liver.

Nitrobenzylthioinosine (NBMPR) was employed as a probe of the nucleoside transporters from rat and guinea-pig liver. Purified liver plasma membranes prepared on self-generating Percoll density gradients exhibited 16-fold (rat) and 10-fold (guinea pig) higher [3H]NBMPR-binding activities than in crude liver homogenates (3.69 and 14.7 pmol/mg of protein for rat and guinea-pig liver membranes respectively, and 0.23 and 1.47 pmol/mg of protein for crude liver homogenates respectively). Binding to membranes from both species was saturable (apparent Kd 0.14 and 0.63 nM for rat and guinea-pig membranes respectively) and inhibited by uridine, adenosine, nitrobenzylthioguanosine (NBTGR) and dilazep. Uridine was an apparent competitive inhibitor of high-affinity NBMPR binding to rat membranes (apparent Ki 1.5 mM). There was a marked species difference with respect to dipyridamole inhibition of NBMPR binding (50% inhibition at 0.2 and greater than 100 microM for guinea-pig and rat respectively). These results are consistent with a role of NBMPR-binding proteins in liver nucleoside transport. Exposure of rat and guinea pig membranes to high-intensity u.v. light in the presence of [3H]NBMPR resulted in the selective radio-labelling of membrane proteins which migrated on sodium dodecyl sulphate/polyacrylamide gels with apparent Mr values in the same range as that of the human erythrocyte nucleoside transporter (45 000-66 000). Covalent labelling of these proteins was abolished when photolysis was performed in the presence of non-radio-active NBTGR as competing ligand.

Affinity Labels↗
PubMed1984

Purification and photoaffinity labelling of lipid methyltransferase from rat liver.

An enzyme that catalyses the three-step methylation of phosphatidylethanolamine to phosphatidylcholine as well as the methylation of fatty acids and that uses S-adenosylmethionine as the methyl donor has been purified about 200-fold from rat liver. Irradiation of the purified enzyme with a short-wavelength u.v. light in the presence of [methyl-3H]8-azido-S-adenosylmethionine followed by electrophoresis results in the incorporation of radioactivity into a single protein band of about 25 kDa. It is concluded that a single catalytic subunit catalyses the conversion of phosphatidylethanolamine into phosphatidylcholine and fatty acid methylation.

Affinity Labels↗
PubMed1985

Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane.

Porcine thyrotropin (TSH) receptors have been purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labelled photoactive derivative (N-hydroxysuccinimidyl 4-azidobenzoate; HSAB) of TSH (125I-HSAB-TSH). Purification of the crosslinked complexes on Sephacryl S-300 followed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that the receptor contained two subunits. One subunit (A) with Mr 45 000 was crosslinked to TSH and the other (B) subunit, Mr 25 000, was linked to the A subunit by a disulphide bridge(s). Other, as yet unidentified, subunits may have been non-covalently associated with the A and B subunits. Analysis of reduced and non-reduced crosslinked TSH receptor-125I-HSAB-TSH on Sephacryl S-300 in the presence and absence of detergent indicated that the A subunit was a hydrophilic peptide. This was confirmed in studies of the release into aqueous solution by reducing agent treatment of 125I-HSAB-TSH crosslinked to the TSH receptor A subunit in thyroid membranes. Similar results were obtained with TSH receptors in human thyroid and guinea pig fat cell membranes. These studies suggest that the hydrophilic A subunit of the receptor forms a binding site for TSH on the outside surface of the cell membrane and that the A subunit is linked to the cell membrane by way of a disulphide bridge to the receptor B subunit.

Affinity Labels↗