Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Photoaffinity Labels”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,225 records · Page 68Linked to original sources
PubMed1982

Use of un-derivatized thyroid hormones for photoaffinity labeling of binding proteins.

Irradiation of the thyroid hormones thyroxine and 3,5,3'-triiodothyronine in the near UV (greater than 300 nm) causes homolytic fission of C--I bonds in both rings. In the presence of hormone-binding proteins, the phenyl radical thus formed, and possibly also the iodine radical, can establish a covalent bond with certain amino acid residues in the binding site. Most if not all of the iodine radicals, however, appear to be reduced to iodide. Incubation of purified carrier proteins for the thyroid hormones in human serum as well as of an extract of rat liver nuclei or of whole nuclei with trace amounts of 125I- or 14C-labeled hormone, followed by irradiation, resulted in covalent binding. This was proven by gel filtration after boiling with guanidine:HCl and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the irradiated solutions or of the excluded-peak material obtained after gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of 125I-labeled irradiated nuclear extracts showed a prominent peak (Mr approximately equal to 45,000), sometimes with a shoulder or small peak at Mr approximately equal to 56,000, and a fast-moving peak (Mr approximately equal to 12,000). Similar patterns were obtained with N-bromoacetylthyroxine or N-bromoacetyltriiodothyronine without irradiation. When a suspension of whole nuclei was irradiated instead of nuclear extracts, the shoulder also became a prominent peak.

Affinity Labels↗
PubMed1982

Photoaffinity labeling with 2-azidoadenosine diphosphate of a tight nucleotide binding site on chloroplast coupling factor 1.

An analog of ADP containing an azido group at the C-2 position of the purine ring has been synthesized and used as an affinity probe of the membrane-bound coupling factor 1 of spinach chloroplast thylakoid membranes. The 2-azido-ADP inhibited light-induced dark binding of ADP at the tight nucleotide binding site on the thylakoid membranes. The 2-azido-ADP itself bound tightly to the thylakoid membranes, with 1 muM as the concentration giving 50% maximum binding. Tight binding of the analog required the thylakoid membranes to be energized, and the nucleotide remained bound after repeated washings of the membranes. The maximum extent of tight binding of the analog (1,2-1.3 nmol/mg of chlorophyll) was stoichiometric with the known coupling factor 1 content of thylakoid membranes but somewhat higher than that observed for ADP (0.5-0.9 nmol per mg of chlorophyll). Tight binding of 2-azido-ADP was decreased by the simultaneous addition of ADP. UV photolysis of washed thylakoid membranes containing tightly-bound 2-azido-[beta-(32)P]ADP resulted in the covalent incorporation of label into the membranes. Isolation of the chloroplast coupling factor 1 from these membranes followed by NaDodSO(4) gel electrophoresis demonstrated that the analog was covalently bound to the beta subunit of the coupling factor complex.

Journal Article↗
PubMed1983

Photoaffinity labeling of the beta-adrenergic receptor from cultured lymphoma cells with [125I]iodoazidobenzylpindolol: loss of the label with desensitization.

The beta-adrenergic antagonist [125I]iodoazidobenzylpindolol ( [125I]IABP) specifically photolabeled two polypeptides in membrane preparations from wild-type (WT) and coupling protein-deficient cyc- cultured lymphoma cells. The molecular weights of the two polypeptides determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis were 65,000 and 55,000. They were labeled in a ratio of approximately 1:1. Pretreatment of intact WT or cyc- cells with 1.0 microM epinephrine for 15 min (desensitization) resulted in a greater loss of the 55,000 Mr polypeptide (40-60%) relative to the 65,000 Mr peptide (10-30% loss). An 18- to 24-hr pretreatment of WT cells with terbutaline (down-regulation) led to a greater than 90% reduction of the photolabeling of both polypeptides, whereas a similar pretreatment of cyc- cells resulted in no further loss of labeled receptor than that observed after only a 15-min pretreatment with epinephrine. There was no indication of a change in the electrophoretic mobility of the [125I]IABP-labeled receptors after either short- or long-term agonist pretreatment. These data provide direct evidence for heterogeneity of the beta-adrenergic receptor in lymphoma cells. The differential loss of the [125I]IABP labeling in the two polypeptides suggests a functional heterogeneity as well.

Adenylyl Cyclases↗
PubMed1984

Highly selective photoaffinity labeling of mu and delta opioid receptors.

We report the synthesis and photolabeling properties of two highly selective ligands for mu and delta opioid-binding sites: Tyr-D-Ala-Gly-MePhe (pN3)-Gly-ol (AZ-DAMGE) and Tyr-D-Thr-Gly-Phe (pN3)-Leu-Thr (AZ-DTLET). An irreversible inhibition of the electrically induced contractions of mouse vas deferens is caused by irradiation (at 254 nm) of the muscle strip in the presence of AZ-DTLET (1 nM). This phenomenon is antagonized only at large concentrations (10 microM) of naloxone, in accordance with the well-known lower selectivity of naloxone for delta sites. Competition experiments with [3H]DAMGE and [3H]DTLET on crude rat brain membranes showed that the azido photoprobes display a similar (AZ-DAMGE) and even a better (AZ-DTLET) selectivity than their respective parent compounds DAMGE and DTLET. Up to 25 nM, AZ-DTLET irreversibly and selectively photolabels the delta sites of crude rat brain homogenates. Due to its lower affinity AZ-DAMGE provides similar selective photolabeling of the mu sites but at higher concentrations (approximately equal to 0.3 microM). When [3H]DAMGE and [3H]DTLET were used as ligands for mu and delta binding subtypes, respectively, no important change in binding capacity and affinity of one receptor type was observed after photolabeling of the other.

Affinity Labels↗
PubMed1985

Photoaffinity labeling of diphtheria toxin fragment A with NAD: structure of the photoproduct at position 148.

Irradiation of mixtures of diphtheria toxin fragment A and [carbonyl-14C]NAD with UV light (253.7 nm) is known to induce efficient transfer of the radiolabel to position 148, corresponding to glutamic acid in the unmodified protein. Here we report the structure of the photoproduct at position 148, as determined by chemical and photochemical methods, fast-atom-bombardment mass spectrometry, and nuclear magnetic resonance. The photoproduct [an alpha-amino-gamma-(6-nicotin-amidyl)butyric acid residue] contains the entire nicotinamide moiety of NAD linked via its number 6 carbon to the decarboxylated gamma-methylene carbon of Glu-148. No portion of the ADP-ribosyl group of NAD is present. These findings are consistent with the idea that Glu-148 lies at or near the catalytic center of diphtheria toxin.

ADP Ribose Transferases↗
PubMed1986

Membrane vesicles from multidrug-resistant human cancer cells contain a specific 150- to 170-kDa protein detected by photoaffinity labeling.

Multiple drug resistance of tumor cells is a common problem in cancer therapy. We have demonstrated that membrane vesicles from highly multidrug-resistant human KB carcinoma cell lines exhibit increased specific and saturable binding of vinblastine. To identify the molecules that bind vinblastine, membrane vesicles from multidrug-resistant cells were exposed to two analogs of vinblastine, N-(p-azido-[3,5-3H]benzoyl)-N'-(beta-aminoethyl)vindesine and N-(p-azido-[3-125I]salicyl)-N'-(beta-aminoethyl)vindesine, that could be photoactivated. Our studies show the specific labeling of a 150- to 170-kDa protein in membrane vesicles from two independently selected multidrug-resistant KB cell lines, which was not seen in drug-sensitive parental or revertant cell lines. The labeling of the high molecular weight protein was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at about 1 microM. Photolabeling was also inhibited by 100 microM vincristine or 100 microM verapamil but not by 100 microM colchicine or 100 microM dexamethasone. The data suggest that the 150- to 170-kDa protein may play an important role in the multidrug-resistance phenotype.

Affinity Labels↗
PubMed1986

Photoaffinity labeling demonstrates binding between Ia molecules and nominal antigen on antigen-presenting cells.

We have used radioiodinated photoreactive bovine insulin as antigen to examine the molecular nature of immunogenic complexes that form on antigen-presenting cells. The probe was allowed to bind to either insulin-presenting B-hybridoma cells, lipopolysaccharide-stimulated blasts, or bovine insulin-specific helper-T-hybridoma cells in the dark. Samples were then exposed to light to induce crosslinkage, solubilized, and analyzed by gel electrophoresis. Two protein bands at about 36 kDa and 27 kDa were specifically labeled on antigen-presenting cells but not on helper T cells. Treatment of these bands with dithiothreitol or endo-beta-N-acetylglucosaminidase F showed that each is composed of a single glycoprotein. These proteins are immunoprecipitable with haplotype-specific but not control anti-Ia antibodies. This identifies the labeled bands as the alpha and beta subunits of class II major histocompatibility antigens. We conclude that a molecular complex may form between Ia and antigen on antigen-presenting cells and that formation of this complex does not require the presence of a helper-T-cell antigen receptor.

Affinity Labels↗
PubMed1986

Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog.

An ADP analog carrying a biotin moiety and a photoreactive group was synthesized. In the presence of vanadate ion (Vi), the analog was tightly trapped into the ATPase site of heavy meromyosin (HMM) or myosin subfragment 1 (S1) in an ADP analog/ATPase site molar ratio of 1:1. UV illumination on the HMM (or S1)-Vi-ADP analog complex resulted in covalent incorporation of the analog into the ATPase site. About 15% of the trapped analog was crosslinked to HMM or S1. Mapping of the crosslinking site of the analog showed that the N-terminal Mr 25,000 segment of the heavy chain participated in binding the ADP analog. The biotin moiety of the analog covalently incorporated into the ATPase site was visualized in electron microscopy by attaching an avidin oligomer. Rotary-shadowed images of the HMM-avidin complex revealed that the crosslinked ADP analog was located about 140 A from the head-rod junction on the head. The result indicates that the ATPase site of myosin is about 140 A apart from the head-rod junction along the head.

Adenosine Diphosphate↗
PubMed1987

Adenine nucleotide binding sites on beef heart F1 ATPase: photoaffinity labeling of beta-subunit Tyr-368 at a noncatalytic site and beta Tyr-345 at a catalytic site.

2-Azidoadenine [32P]nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F1 ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the beta subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major 32P-labeled tryptic fragments showed beta-subunit Tyr-368 to be present at noncatalytic sites and beta Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.

Adenine Nucleotides↗
PubMed1989

Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein.

An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with the modified p21 protein, the cells were pulsed with [35S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21-protein complexes. By using this technique, we found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein into the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. We suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes.

Affinity Labels↗
PubMed1991

Direct photoaffinity labeling of tubulin with colchicine.

Ultraviolet irradiation of the [3H]colchicine-tubulin complex leads to direct photolabeling of tubulin with low but practicable efficiency. The bulk (70% to greater than 90%) of the labeling occurs on beta-tubulin and appears early after irradiation, whereas alpha-tubulin is labeled later. The labeling ratio of beta-tubulin to alpha-tubulin (beta/alpha ratio) is reduced by prolonged incubation, prolonged irradiation, urea, high ionic strength, the use of aged tubulin, dilution of tubulin, or large concentrations of colchicine or podophyllotoxin. Glycerol increases the beta/alpha ratio. Limited data with [3H]podophyllotoxin show that it covalently bound with a similar beta/alpha distribution. Vinblastine, on the other hand, exhibits preferential attachment to alpha-tubulin. The possibilities that colchicine binds at the interface between alpha-tubulin and beta-tubulin, that the drug spans this interface, and that both subunits may contribute to the binding site are suggested.

Affinity Labels↗
PubMed1992

An auxin-binding protein is localized to the plasma membrane of maize coleoptile cells: identification by photoaffinity labeling and purification of a 23-kda polypeptide.

Plasma membrane vesicles were isolated from maize (Zea mays L.) coleoptile tissue by aqueous two-phase partitioning and assayed for homogeneity by the use of membrane-specific enzymatic assays. Using 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA), we identified several IAA-binding proteins with molecular masses of 60 kDa (pm60), 58 kDa (pm58), and 23 kDa (pm23). Using Triton X-114, we were able to selectively extract pm23 from the plasma membrane. We show that auxins and functional analogues compete with [3H]N3IAA for binding to pm23. We found that PAB130, a polyclonal antibody raised against auxin-binding protein 1 (ABP-1), recognized ABP-1 as well as pm23. This suggests that pm23 shares common epitopes with ABP-1. In addition, we identified an auxin-binding protein with a molecular mass of 24 kDa (pm24), which was detected in microsomal but not in plasma membrane vesicle preparations. Like pm23 this protein was extracted from membrane vesicles with Triton X-114. We designed a purification scheme allowing simultaneous purification of pm23 and pm24. Homogeneous pm23 and pm24 were obtained from coleoptile extracts after 7000-fold purification.

Journal Article↗
PubMed1992

Photoaffinity labeling of the primary fibrin polymerization site: localization of the label to gamma-chain Tyr-363.

Fragment D prepared from human fibrinogen was labeled specifically by photoactivation of the peptide [14C]Gly-Pro-Arg-N-(4-azido-2-nitrophenyl)Lys amide. The preparation was freed of excess labeling reagents and then reduced and alkylated. The component alpha, beta, and gamma chains were purified by chromatography on carboxymethylcellulose and the radioactivity was found to be restricted to the gamma chain. Isolated gamma chains were digested with various endopeptidases, both alone and in tandem, and the products were fractionated by gradient HPLC. The amino acid compositions of all labeled peptides led to the conclusion that the modification occurs exclusively on gamma-chain Tyr-363.

Affinity Labels↗
PubMed1995

Photoaffinity labeling of a peptide substrate to myosin light chain kinase.

The substrate binding properties of skeletal muscle myosin light chain kinase were investigated with a synthetic peptide containing the photoreactive amino acid p-benzoylphenylalanine (Bpa) incorporated amino-terminal of the phosphoacceptor serine (BpaKKRAARATSNVFA). When photolyzed at 350 nm, the peptide was cross-linked stoichiometrically to myosin light chain kinase in a Ca2+/calmodulin-dependent manner. Peptide incorporation into kinase inhibited light chain phosphorylation, and the loss of kinase activity was proportional to the extent of peptide incorporated. After peptide I was incorporated into myosin light chain kinase, it was partially phosphorylated in the absence of Ca2+/calmodulin. The extent of phosphorylation increased in the presence of Ca2+/calmodulin. The cross-linked photoadduct was digested, labeled peptides were purified by high performance liquid chromatography, and sites of covalent modification were determined by amino acid sequencing and analysis. The covalent modification in the catalytic core occurred on Ile-373 (66%) and in a peptide containing residues Asn-422 to Met-437 (14%), respectively. Lys-572 in the autoinhibitory region accounted for 20% of the incorporated label. The coincident covalent modification of the autoinhibitory domain suggests that it is located near the catalytic site. Based upon a model of the catalytic core, the substrate peptide is predicted to bind in the cleft between the two lobes of the kinase. The orientation of the substrate peptide on myosin light chain kinase is similar to the orientation of the substrate recognition fragment, but not the high affinity binding fragment, of inhibitor peptide of cAMP-dependent protein kinase in the catalytic subunit of the cAMP-dependent protein kinase.

Adenosine Triphosphate↗
PubMed1996

Definition of a nucleotide binding site on cytochrome c by photoaffinity labeling.

We have used TNP-8N3-AMP (2'(3')-O-(2,4,6-trinitrophenyl)-8-azidoadenosine monophosphate) and TNP-8N3-ATP to probe the ATP binding site(s) of cytochrome c. Irradiation of cytochrome c with close to stoichiometric amounts of TNP-8N3-AMP at low ionic strength derivatized approximately half of the protein, with the mono-derivatized species being associated with four peaks (B, 6%; C, 17%; D, 24%; E, 4%) eluted from a cation exchange column. Irradiation in the presence of ATP suggested that the main peaks C and D resulted from more specific nucleotide binding. Thermolysin digestion and TNP-peptide purification and sequencing revealed that peak C was associated with derivatization of mainly Lys-86 and to a lesser extent Lys-72 and peak D with mainly Lys-87 and less so with Lys-72. Minor peaks B and E could not be identified. TNP-8N3-ATP photolabeling produced similar results, showing favored interaction of the adenyl ring with Lys-86 and Lys-87 and to a lesser extent with Lys-72. The results are compatible with previous findings that suggest that the principal locus of ATP binding is at nearby Arg-91 (Corthesy, B. E., and Wallace, C. J. A.(1986) Biochem. J. 236, 359-364). Molecular modeling with energy-minimized docking of ATP between the 60s helix and the 80s stretch with the gamma-phosphate constrained to interact with Arg-91, places the 8 position close to Lys-86 and Lys-87 in the anti conformation about the glycosidic bond and to Lys-72 in the syn conformation, and the ribose hydroxyls within H-bonding distance of Glu-69.

Adenosine Monophosphate↗
PubMed1998

Direct photoaffinity labeling of cysteine-295 of alpha-tubulin by guanosine 5'-triphosphate bound in the nonexchangeable site.

The alphabeta-tubulin heterodimer has two high affinity guanosine 5'-triphosphate binding sites, so that purified tubulin usually contains two molecules of bound guanosine nucleotide. Half this nucleotide is freely exchangeable with exogenous guanine nucleotide, and its binding site has been readily localized to the beta-subunit. The remaining nonexchangeable guanosine 5'-triphosphate can only be released from tubulin by denaturing the protein. We replaced the exchangeable site nucleotide of tubulin with 2'-deoxyguanosine 5'-diphosphate, exposed the resulting tubulin to ultraviolet light, degraded the protein, and isolated ribose-containing peptide derived from the nonexchangeable site. A large cyanogen bromide peptide was recovered, and its further degradation with endoproteinase Glu-C established that cysteine-295 of alpha-tubulin was the major reactive amino acid cross-linked to guanosine by ultraviolet irradiation.

Affinity Labels↗
PubMed2002

Involvement of the second extracellular loop (E2) of the neurokinin-1 receptor in the binding of substance P. Photoaffinity labeling and modeling studies.

Substance P (SP) interacts with the neurokinin-1 (NK-1) G-protein-coupled receptor, which has been cloned in several species. In the present study, the domains of the NK-1 receptor involved in the binding of SP and SP-(7-11) C-terminal fragment have been analyzed using two peptide analogs containing the photoreactive amino acid para-benzoylphenylalanine ((p-Bz)Phe) in position 8 of their sequence. This study was carried out with [BAPA-Lys(6),(p-Bz)Phe(8),Pro(9),Met(O(2))(11)]SP-(7-11) and [BAPA(0),(p-Bz)Phe(8)]SP on both rat and human NK-1 receptors expressed in CHO cells. Combined trypsin and endo-GluC enzymatic complete digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis led to the identification of the same domain of covalent interaction, (173)TMPSR(177), for the two photoactivatable peptides. Further digestion of this fragment with carboxypeptidase Y led to the identification of (173)TMP(175) in the second extracellular loop (E2) of the NK-1 receptor as the site of covalent attachment. Models of the conformation of this E2 loop in the human NK-1 receptor were generated using two different strategies, one based on homology with bovine rhodopsin and the other based on the solution conformation preferences of a synthetic peptide corresponding to the E2 loop.

Amino Acid Sequence↗
PubMed1995

Photoaffinity labeling of influenza virus RNA polymerase PB1 subunit with 8-azido GTP.

8-Azido GTP (8-N3 GTP) was demonstrated to be polymerized into RNA by influenza virus-associated RNA polymerase at about one tenth the rate of GTP incorporation. The Km value for the azido analogue of GTP in primer-dependent RNA synthesis was 94 microM whereas Km for the natural substrate, GTP, was 6.7 microM. Upon exposure of a mixture of 8-N3 [alpha-32P]GTP and influenza virus ribonucleoprotein (RNP) complexes to ultraviolet light, the PB1 subunit of viral RNA polymerase was selectively radio-labeled. The photo-labeling of PB1 was competed strongly by GTP and to lesser extents by other nucleoside 5'-triphosphates. These results altogether support the prediction that the substrate-binding site (S site) of influenza RNA polymerase is located on the PB1 protein. In the presence of ApG primer, the 8-N3 GTP binding was reduced to about 40% level, suggesting that the GTP analogue can bind not only to the S site but also to the primer- and product-binding site (P site).

Affinity Labels↗