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PubMed1987

Photoaffinity labelling of the TSH receptor on FRTL5 cells.

An investigation of the properties of TSH receptors on FRTL5 cells using affinity labelling with a 125I-labelled photoactive derivative of TSH is described. Our studies suggest that FRTL5 cells contain 2 principal types of cell surface TSH receptors. One form, probably a precursor, consists of a single polypeptide chain (Mr 120,000) with an intrachain loop of amino acids formed by a disulphide bridge. The other type of receptor consists of a water-soluble A chain (Mr 55,000) linked to an amphiphilic B chain (Mr 35,000) by a disulphide bridge. The 2 chain structure is probably derived from the single chain 120,000 protein by enzymatic cleavage of peptide sequences within the loop of amino acids formed by the intrachain disulphide bridge.

Affinity Labels↗
PubMed1987

Catalytic and noncatalytic nucleotide binding sites of chloroplast F1 ATPase. Photoaffinity labeling and peptide sequencing.

Exposure of chloroplast F1 ATPase to 2-azido-ATP results in the noncovalent tight binding of 2-azido-ATP or 2-azido-ADP to noncatalytic or to catalytic sites. Subsequent photolysis results in covalent labeling of adjacent tryptic peptides of the beta-subunit. Binding at noncatalytic sites results in labeling of tyrosine 385 by an ATP or an ADP moiety. Binding at catalytic sites results in labeling of tyrosine 362 by only an ADP moiety. Similar labeling patterns are observed for the heat-activated or the membrane-bound enzymes.

Adenine Nucleotides↗
PubMed1987

Photoaffinity labelling of melanoma cell MSH receptors.

UV-irradiation at 365 nm of cultured Cloudman S91 mouse melanoma cells in the presence of photoreactive alpha-MSH analogues induced longlasting receptor stimulation as revealed by the ensuring activation of tyrosinase. Receptor labelling was more efficient with 4-diazirinophenyl and 2-nitro-4-azidophenyl photolabels than with 4-azidophenyl, and was further increased when superpotent [Nle4,D-Phe7]-alpha-MSH was used as ligand. Incubation of B16 melanoma cell membranes with mono-iodinated [Nle4,D-Phe7,Trp-(Naps)9]-alpha-MSH followed by UV-irradiation at 310-550 nm labelled a single band on SDS-PAGE with a molecular mass approximately or equal to 45 kDa. The displacement curve obtained in a competitive photolabelling experiment paralleled that of the binding assay, demonstrating that the labelling was specific.

Affinity Labels↗
PubMed1987

A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets.

A photoactive iodoarylazide derivative (I-APA-PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor-binding studies using [3H]U46619, I-APA-PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I-APA-PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding ratio-iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2-binding protein in human platelets.

Affinity Labels↗
PubMed1988

Photoaffinity labelling shows that Escherichia coli isocitrate dehydrogenase kinase/phosphatase contains a single ATP-binding site.

Ultraviolet irradiation of E.coli isocitrate dehydrogenase kinase/phosphatase in the presence of 8-azidoATP resulted in parallel losses of its kinase and phosphatase activities, and in covalent attachment of the reagent to the protein at a single site. ATP and ADP protected the two activities to similar extents. The data suggest that the activation of the phosphatase by adenine nucleotides results from binding of the nucleotides to the active site of the kinase.

Adenosine Diphosphate↗
PubMed1988

Photoaffinity labeling of a 33 kDa protein subunit of the delta-opioid receptor in neuroblastoma and hybrid cell lines.

Tritiated DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) binds with high affinity, specificity and saturability to neuroblastoma N18TG2 and hybrid neuroblastoma x glioma NG108-15 and NG108-5 intact cells. The delta-opioid receptor density in cells cultured in chemically defined medium was increased about 2 times compared to that in cells cultured in 10% fetal calf serum. A major and a minor protein species covalently and specifically bound to [125I]azido-DTLET (Tyr-D-Thr-Gly-pN3Phe-Leu-Thr), photoactivatable ligand, migrated on SDS-gel electrophoresis with Mr values near 33,000 and 58,000, respectively.

Affinity Labels↗
PubMed1989

Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes.

2-Azidoadenosine was synthesized from 2-chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2-azidoadenosine 3',5'-bisphosphate. The bisphosphate was labeled in the 5'-position using the exchange reaction catalyzed by T4 polynucleotide kinase in the presence of [gamma-32P]ATP. Polynucleotide kinase from a T4 mutant which lacks 3'-phosphatase activity (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2-Azidoadenosine 3',5'-[5'-32P]bisphosphate can serve as an efficient donor in the T4 RNA ligase reaction and can replace the 3'-terminal adenosine of yeast tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross-linked to the Escherichia coli ribosome.

Adenosine Diphosphate↗
PubMed1992

Photoaffinity labeling of DNA polymerase alpha DNA primase complex based on the catalytic competence of a dNTP reactive analog.

FABdCTP was found to be a substrate of DNA polymerization catalyzed by a DNA polymerase alpha-DNA primase complex on the 5'-GTGAGTAAGTGGAGTTTGGCACGAT-3' template and 3'-CTCAAACCGT-5' primer. After complete primer extension in the presence of FABdCTP under UV-irradiation of the reaction mixture, 70% of the template was covalently linked to the primer. Labeling of the 165 kDa subunit of the DNA polymerase alpha, 59 kDa and 49 kDa subunits of the DNA primase and an unknown protein with apparent molecular weight of 31 kDa was observed. By another way of protein labeling FABdCTP was covalently bound to the subunits of the enzyme under UV irradiation and then this moiety was introduced into the 3'-end of the 5'-[32P]primer by the catalytic activity of DNA polymerase or DNA primase. In this case covalent labeling of the 165 kDa, 49 kDa and 31 kDa subunits was observed.

Affinity Labels↗
PubMed1992

Characterization of myocardial extracellular ATP receptors by photoaffinity labelling and functional assays.

Extracellular ATP receptors in rat ventricular myocytes were investigated through intact cell photolabelling followed by protein isolation. 8-Azido-ATP (8Az-ATP) was used for labelling under specific conditions determined by parallel functional studies. In those studies ATP-induced cytosolic Ca2+ transients were irreversibly and specifically inhibited by UV-photolyzed 8Az-ATP, but not by 2-azido-ATP (2Az-ATP), even in the presence of high concentrations of phosphonucleotides not affecting myocardial ATP receptors. Under those conditions background labelling is minimized and radioactive 8Az-ATP specifically labels a band of 45-48 kDa on a SDS gel. Labelling under the above conditions in the presence of ATP gamma S or 2-methylthio-ATP (2-meSATP), which are distinct for two functionally different cardiac ATP receptors, shows two different proteins within the same band consistent with the possible labelling of these two receptors.

Adenosine Diphosphate↗
PubMed1995

Photoaffinity labeling of mitochondrial proteins with 2-azido [32P]palmitoyl CoA.

A long-chain fatty acyl CoA photolabel, 2-azido [32P]palmitoyl CoA, was synthesized and its covalent interaction with mitochondrial membrane proteins examined. On binding of 2-azido [32P]palmitoyl CoA to beef heart mitochondria, two polypeptides were primarily labeled, the 30 kDa ADP/ATP carrier and a 41 kDa protein of unknown identity. Carboxyatractyloside and palmitoyl CoA completely protected against labeling of the 30 kDa protein indicating that it was the ADP/ATP carrier. With inverted submitochondrial particles, only the 30 kDa polypeptide was labeled by 2-azido [32P]palmitoyl CoA. The labeling was inhibited by bongkrekic acid and palmitoyl CoA but not carboxyatractyloside, providing evidence that the ADP/ATP carrier was covalently bound from the matrix side of the membrane. In brown adipose tissue mitochondria, 2-azido [32P]palmitoyl CoA photolabeled the ADP/ATP carrier and the 32 kDa uncoupling protein with some minor labeling of 36 and 68 kDa polypeptides. The results indicated that this physiological photolabeling reagent with the azido group on the CoA portion of the molecule interacts like 2-azido ADP with nucleotide binding sites of a number of important enzymes in cell metabolism. Moreover, the evidence strongly supports the hypothesis that long chain fatty acyl CoA esters are natural ligands for key nucleotide binding proteins.

Acyl Coenzyme A↗
PubMed1996

Photoaffinity labelling of the mitochondrial uncoupling protein by [3H]azido fatty acid affects the anion channel.

Brown adipose tissue (BAT) mitochondria were incubated with the azido derivative of fatty acid (hexadecanoic) containing four tritium atoms, [3H]AzHA, and among all mitochondrial proteins only a few proteins were photolabelled after irradiation with UV. It suggests the existence of specific fatty acid binding sites on mitochondrial proteins. It was also possible to label with [3H]AzHA the isolated uncoupling protein (UcP) of BAT mitochondria with a low stoichiometry--lower than one AzHA per dimeric UcP. These results together with the observed competition (i.e. prevention of photolabelling) of various UcP anionic substrates with [3H]AzHA and its dodecanoic acid analogue, suggest the existence of the specific fatty acid binding site on UcP identical with the anion channel or anion translocating site.

Adipose Tissue, Brown↗
PubMed1996

Specific photoaffinity labelling of a ferripyoverdin outer membrane receptor of Pseudomonas aeruginosa.

In order to identify and characterize the receptors involved in pyoverdin-mediated iron transport in Pseudomonas aeruginosa ATCC 15692, a photoactivatable siderophore has been synthesized. In the dark, this probe is stable and is able to promote iron transport at the same rate as the native pyoverdin. Under irradiation at 312 nm, the molecule is photodecomposed and a clear inhibition of the iron transport is observed. With the radioactive form of this photoactivatable probe, we were able to visualize on a SDS-PAGE gel a labelled protein of approximately 90 kDa molecular mass, which is very likely the FpvA receptor or a yet unknown pyoverdin receptor.

Affinity Labels↗
PubMed1993

Solubilization, characterization and photoaffinity labeling of the mitochondrial dihydropyridine receptor from bovine adrenal medulla.

1. The mitochondrial dihydropyridine receptor was solubilized with Chaps at a detergent/protein ratio of 2.5, during 45 min at 4 degrees C. 2. From the rate constants of association (8.10 +/- 0.25 x 10(4) M-1 min-1) and dissociation (0.022 +/- 0.001 min-1) a Kd of 275 nM was calculated, while from saturation experiments a Kd of 270 +/- 30 nM and a density of receptors of 106 +/- 9 pmol/mg protein was obtained. 4. The solubilized receptors are heat-resistant, sensitive to the trypsin and to the reduction of disulfide bonds. 5. In native membranes, a polypeptide of 50 kDa was specifically photolabelled with [3H]Azidopine.

Adrenal Medulla↗
PubMed1988

Photoaffinity labelling with [3H]R1881 of androgen receptors from human cultured genital fibroblasts of normal individuals and patients with androgen receptor abnormalities.

Androgen receptors were partially purified by affinity chromatography of cytosols prepared from cultured genital fibroblasts of normal individuals and patients with androgen receptor abnormalities. Partially purified receptors were covalently labelled with [3H]R1881 (triated methyltrienolone) by ultraviolet photoactivation. Gel electrophoresis of cytosols from normal individuals showed a single radioactive peak, Mr approximately 97 K. Cytosols from patients with decreased nuclear transfer showed a similar peak, Mr approximately 97 K; cytosols from patients with partial androgen insensitivity (PAIS) or receptor positive complete androgen insensitivity (CAIS, R+) showed a peak Mr approximately 30 K-43 K.

Affinity Labels↗