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PubMed1980

Specific photoaffinity labeling induced by energy transfer: application to irreversible inhibition of acetylcholinesterase.

p-Dimethylaminobenzene diazonium fluoroborate belongs to a class of potential photoaffinity labeling reagents which, by irradiation, produces a highly reactive electrophilic species. In addition, it can be photodecomposed by photoexcited tryptophan derivatives (e.g., N-acetyltryptophanamide and tryptophan residues belonging to acetylcholinesterase) by an energy transfer reaction. This substance is a competitive inhibitor of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) and is able to inactivate the enzyme either by photoaffinity labeling after irradiation at 410 nm or by an energy transfer reaction after irradiation at 295 nm. The efficiency of this method is demonstrated by an increase of the rate of enzyme inactivation as well as by a decrease of nonselective labeling with a radioactive inhibitor p-[methyl-3H]-dimethylaminobenzene diazonium fluoroborate.

Affinity Labels↗
PubMed1998

Photoaffinity labeling of rat liver glutathione S-transferase, 4-4, by glutathionyl S-[4-(succinimidyl)-benzophenone].

Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and xenobiotic substrate, was synthesized and shown to act as a photoaffinity label of rat liver glutathione S-transferase, 4-4. A time-dependent photoinactivation occurs upon irradiation at long wavelength UV light of the complex of enzyme and GS-Succ-BP. The rate of inactivation exhibits nonlinear dependence on [GS-Succ-BP], characterized by an apparent KI of 115 microM and kmax of 0.469 min-1. Effective protection against photoinactivation by 150 microM GS-Succ-BP is provided by dinitrophenol, nitrobenzene, ethacrynic acid, and S-hexylglutathione, analogues of xenobiotic substrates and product. These results suggest that GS-Succ-BP reacts with the enzyme within the active site, probably in the xenobiotic substrate-binding site. Upon complete inactivation, reagent incorporation of about 1 mol/mol of enzyme dimer is measured by radioactivity and MALDI-TOF mass spectrometry. Isolation of modified peptides followed by gas-phase sequencing and mass spectrometry indicates that Met-112 is the only reaction target of GS-Succ-BP. Although only one subunit of the enzyme dimer is modified, catalytic activity of both subunits is lost. Molecular modeling suggests that the benzophenone moiety of the compound binds in the cleft between the two enzyme subunits and modification of Met-112 on one subunit excludes reaction of the corresponding methionine on the other subunit. It is proposed that the new compound, glutathionyl S-[4-(succinimidyl)benzophenone], may have general applicability as a photoaffinity label of other enzymes with glutathione binding sites.

Animals↗
PubMed1984

Interaction of purines and related compounds with photoaffinity-labelled benzodiazepine receptors in rat brain membranes.

The interaction of purine-receptor agonists and antagonists with [3H]Ro15-1788 binding sites in rat brain membranes was examined before and after UV-photoaffinity labelling of a proportion of the sites with flunitrazepam. Whereas photoaffinity labelling of the receptors reduced benzodiazepine agonist affinity but not benzodiazepine antagonist affinity, the IC50S of adenosine-receptor agonists, partial agonists and antagonists were unaltered by the conformational changes in the benzodiazepine receptors which are thought to be induced by the photolabelling process. The affinity of dipyridamole, a potent adenosine uptake blocker and potent displacer of [3H]diazepam binding, was drastically reduced by photolabelling.

Affinity Labels↗
PubMed1989

The ADP/ATP carrier from yeast (AAC-2) is uniquely suited for the assignment of the binding center by photoaffinity labeling.

The ADP/ATP carrier from yeast was photoaffinity-labeled in mitochondria with 2-azido-[alpha-32P]ATP in a binding-center-specific, i.e. carboxyatractylate-sensitive, manner. After isolation, fragmentation possibilities unique for the yeast AAC-2 could be exploited to assign the insertion to a narrow range of the sequence. The CNBr fragment 115-210 contained all the incorporated label which corresponds to the second domain within the triple-domain primary structure of the AAC. With hydroxylamine cleavage directed to the Asn 171-Gly 172 site, all the label was found in the C-terminal 16 kDa fragment. Thus the 2-azido-ATP incorporation is clearly delimited to the 172-210 segment. 8-Azido-[alpha-32P]ATP could be site-specifically incorporated only in isolated AAC since it has a much lower affinity for AAC than 2-azido-ATP. The label was also exclusively found in the 172-210 region. With both forms no incorporation into the C-terminal region was found, as claimed for bovine AAC. The labeled segment contains Lys 179 and 182 which are homologous to bovine Lys 162 and 165 and which have been proposed to be in the translocation path.

Adenosine Triphosphate↗
PubMed1990

Purification and photoaffinity labeling of the I-Ak histocompatibility molecule.

Photoaffinity labeling was used to evaluate optimal conditions for purification of I-A k histocompatibility molecules in functionally active form. We assessed the biological activity of I-A k primarily by its binding of the hen egg-white lysozyme (HEL) peptide from residues 46-61. [125I]iodo,4-azidosalicyloly(HEL)46-61 (IASA-46-61)-labeled I-A k on B cell hybridoma membranes and their detergent solubilisates, at the alpha chain. Following extensive detergent dialysis, the intensity of this labeling remained unchanged in the case of MEGA 8 and MEGA 9 detergents, but decreased in the case of deoxycholate and n-octylglucoside. Conditions for affinity purifications were assessed on one hand by determining the dissociation conditions of I-A k from various monoclonal antibodies and by determining the denaturation of I-A k under these conditions. Effective dissociation in the absence of detectable denaturation was observed for 10.3.6.2 and 40.LH monoclonal antibody at pH 3.5 and to a lesser extent at low concentrations of ammonium thiocyanate and guanidine thiocyanate at neutral pH. I-A k purified from cell membranes using MEGA 8 and MEGA 9 detergent mixtures and acid elution from 10.3.6.2 Sepharose was efficiently labeled by IASA-46-61. Thus I-A k was active in antigen presentation to a T cell hybridoma when reconstituted in planar membranes. In contrast to I-A k on cell membranes, purified I-A k in detergent showed extensive labeling of the beta chain. The overall labeling intensity and the extent of beta chain labeling substantially changed upon addition of certain lysophosphatides.

Affinity Labels↗
PubMed1984

Tautomerism of 2-azidoadenine nucleotides. Effects on enzyme kinetics and photoaffinity labeling.

The 2-azidoadenine nucleotides show promise as photoaffinity probes. Substitution at the C-2 position should favor an anti conformation and enable binding of the analogue to enzyme sites which exhibit low affinity for the 8-azidoadenine derivatives. The 2-azidoadenine nucleotides were found to be substrates for pyruvate kinase, phosphofructokinase, adenylate kinase, hexokinase and the mitochondrial F1-ATPase. However, tautomerism of 2-azidoadenine nucleotides to two nonphotoreactive tetrazole forms complicates kinetic analyses and their use as photoaffinity probes. An analysis of the ultraviolet spectra of these analogues enables an estimation of the tetrazolo isomer content and the rates of tautomerization. The photoreactive azido isomer was found to represent only 45% of the total analogue population in neutral aqueous solution. The azidoazomethine-tetrazole equilibrium favors the azido isomer in acidic or nonpolar solutions. The first-order rate constants at 25 degrees C were determined to be 0.017 min-1 and 0.021 min-1 for tautomerism to the azido and tetrazolo isomers, respectively. Prior equilibration of the probe in various solvents thus allows investigation of the analogue's behavior with an enzyme system at different, essentially fixed, isomer ratios. The determination of the impact of the tetrazolo tautomers on the system allows optimization of conditions for photoaffinity-labeling experiments.

Adenosine Diphosphate↗
PubMed1991

Photoaffinity labeling of soluble auxin-binding proteins.

The photoaffinity labeling agent azido-IAA (5-N3-[7-3H]indole-3-acetic acid), a biologically active analogue of the endogenous auxin indole-3-acetic acid, was used to search for auxin-binding proteins in the soluble fraction of Hyoscyamus muticus cells. Azido-IAA became covalently attached to three polypeptides with a high specific activity. The labeling was specific for IAA and not due to random tagging. Two polypeptides with molecular masses of 31 and 24 kDa in the 0-30% ammonium sulfate fraction were labeled after UV photolysis at 0 degree C but not at -196 degrees C, and appeared to have a high affinity indole-binding site(s) for which active, non-indole auxins were not good ligands. A third polypeptide with a molecular mass of 25 kDa present in the 50-60% ammonium sulfate fraction labeled exclusively at -196 degrees C and had a significant affinity for active auxins but not for inactive indoles. The azido-IAA labeling pattern, pI, competition results, and immunoprecipitation all indicate that the 31- and 24-kDa polypeptides are related to the basic form of endo-1,3-beta-glucanase (EC 3.2.1.39). Azido-IAA labeling polypeptides equivalent to the 31- and 24-kDa species were apparently also present in the cell wall. The low pH optimum for binding of azido-IAA to the 25-kDa polypeptide suggests the location of the active protein in a compartment such as the vacuole or a transport vesicle rather than in the cytosol.

Affinity Labels↗
PubMed1982

Demonstration of specific high affinity binding sites in plasmid DNA by photoaffinity labeling with an ethidium analog.

We have used photoaffinity labeling of pBR322 DNA with 8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride to demonstrate high affinity ethidium-binding sites. Plasmid equilibrated with as little as 1 drug/DNA molecule was photoactivated, freed of uncomplexed drug by ethanol precipitation, and subjected to restriction analysis. There was highly specific, rather than random, blockage of HhaI sites (d(GCGC)) at low drug concentrations. Furthermore, the same 7 new digestion fragments were generated at drug to nucleotide ratios ranging from 1:100 to 1:8000. All the new DNA fragments had chain lengths greater than the largest HhaI fragment (393 base pairs). At higher ligand concentrations closely approximating those needed for equilibrium binding studies, detection of the high affinity sites was greatly masked. Drug binding to HhaI restriction fragments which had been prepared prior to the action of drug did not induce new bands. Furthermore, the larger DNA fragments from drug-labeled plasmid were resistant to HhaI digestion over a wide range of enzyme concentrations. These findings suggest that ligand binding can be highly selective even between sites which have the same tetranucleotide sequence. Therefore, selective drug binding must be dictated not only by local base sequence preference, but also by other long range parameters.

Affinity Labels↗
PubMed1991

Photoaffinity labeling of the 1,25-dihydroxyvitamin D-3 receptor.

Underivatized 1,25-dihydroxy[26,27-3H]vitamin D-3 was successfully used to photoaffinity label the 1,25-dihydroxyvitamin D-3 receptor. The covalent incorporation of tritium into the receptor protein was induced by ultraviolet irradiation of the receptor-1,25-dihydroxy[26,27-3H]vitamin D-3 complex in crude pig intestinal nuclear extract. The amount of incorporated label increased with increasing time of irradiation and was dependent on light of wavelengths 220-280 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography were used to demonstrate that label was incorporated primarily into the 1,25-dihydroxyvitamin D-3 receptor. In addition, the label incorporation was eliminated by competition with a 100-fold excess nonradioactive 1,25-dihydroxyvitamin D-3, indicating that the label was specific for the steroid binding site. Since 1,25-(OH)2[26,27-3H]vitamin D-3 is readily available and requires no special precautions for its preparation and handling, it should be a useful photoaffinity label for future studies of the receptor.

Affinity Labels↗
PubMed1994

Photoaffinity labeling of the ATP binding domain of Rubisco activase and a separate domain involved in the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Photoaffinity labeling of Rubisco activase with 2- and 8-N3ATP was used to identify the adenine binding domain for ATP. Rubisco activase hydrolyzed both of these analogs of ATP and used their hydrolysis to support a low rate of Rubisco activation. When irradiated with ultraviolet light, these and other azido-substituted adenine nucleotides covalently modified Rubisco activase at two distinct binding sites. Competition binding experiments with ATP and ADP showed that one of the sites was the ATP binding domain. The other site was not a nucleotide binding domain per se but would bind adenine nucleotides if an azido moiety was present on the base. Tryptophan and other indoles prevented azidoadenine nucleotides from labeling this domain but afforded little protection to the ATP binding domain. The ability to selectively protect each of the two binding sites made it possible to localize the adenine binding domain for ATP to the region of Rubisco activase from N68-D74 and the other binding domain to a region near the N-terminus from Q10 to D14. Modification of the region from Q10 to D14 by photoaffinity labeling prevented Rubisco activase from promoting activation of Rubisco without affecting ATP hydrolysis. These data suggest that a specific region of Rubisco activase near the N-terminus may be a site of interaction with Rubisco. Binding of azidoadenine nucleotides in this region appears to be fortuitous and may involve base-stacking with the species-invariant Trp at position 16 and hydrogen bonding of the azido moiety.

Adenosine Triphosphate↗
PubMed1986

Vinblastine photoaffinity labeling of a high molecular weight surface membrane glycoprotein specific for multidrug-resistant cells.

Photoactive radioactive analogues of vinblastine were used to photoaffinity label membranes of Chinese hamster lung drug-sensitive (DC-3F), multidrug-resistant sublines selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/ADX), and revertant (DC-3F/ADX-U) cells. A radiolabeled doublet (150-180 kDa) consisting of a major and minor band which was barely detectable in parental drug-sensitive cells was increased up to 150-fold in the drug-resistant variants but only 15-fold in the revertant cells. Photoaffinity labeling in the presence of 200-fold excess vinblastine reduced radiolabeling of the 150-180-kDa species up to 96%, confirming its Vinca alkaloid binding specificity. The radiolabeled doublet comigrated with a Coomassie Blue stained polypeptide doublet in the drug-resistant cells and was immunoprecipitated with polyclonal antibody which is specific for the 150-180-kDa surface membrane glycoprotein in multidrug-resistant cell lines. The identification of this Vinca alkaloid acceptor in multidrug-resistant plasma cell membranes suggests the possibility of a direct functional role for the 150-180-kDa surface membrane protein in the development of multidrug resistance.

Affinity Labels↗
PubMed1974

Photoaffinity labeling of the ouabain-binding site on (Na+ plus K+) adenosinetriphosphatase.

An ethyl diazomalonyl derivative of cymarin was synthesized in order to photoaffinity label the cardiac glycoside-binding site on (Na(+) + K(+)) adenosinetriphosphate (EC 3.6.1.3). When a noncovalent complex of the enzyme and this cardiac glycoside derivative was photolyzed, a covalent bond was formed between the ligand and the larger of the two polypeptide subunits of the enzyme. Several control experiments demonstrate that this photochemical reaction occured while the ligand was bound to the site at which it inhibits the enzyme activity. Another specific inhibitor, tentatively identified as the ethyl chloromalonyl derivative of cymarin, produced similar photoaffinity labeling of the larger subunit, demonstrating that the photolytic dissociation of the diazo group may not be responsible for the photochemical reaction. Since the cardiac glycoside-binding site, which is accessible from the outside surface of the plasma membrane, and the phosphorylation site, which is accessible from the inside surface, are both on the larger polypeptide subunit of (Na(+) + K(+)) adenosinetriphosphatase, this polypeptide has sequences exposed to both sides of the membrane.

Adenosine Triphosphatases↗
PubMed1983

Photoaffinity labeling of the chick progesterone receptor proteins. Similar hormone binding domains detected after removal of proteolytic interference.

Chick progesterone receptor subunits A and B have been photoaffinity-labeled using [3H]R5020 ([17 alpha-methyl-3H]17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione) by a modification of the procedures previously reported by our laboratory (Dure, L. S., IV, Schrader, W. T., and O'Malley, B. W. (1980) Nature (Lond.) 283, 784-786). [3H]R5020 binds to the same receptor sites as authentic progesterone, and has an apparent Kdiss of 2.0 nM. Use of a CuSO4 filter raised the coupling efficiency to 5% and labeled exclusively the receptor proteins. Smaller labeled macromolecules were found to be proteolytic fragments of receptors. The protease(s) could not be inhibited by any of the commonly used protease inhibitors. However, the proteolytic activity was completely removed by passage of crude receptor preparations through phosphocellulose columns. Receptor preparations, photoaffinity-labeled after this procedure, showed exclusively one radioactive band at Mr = 79,000 (subunit A) or Mr = 108,000 (subunit B) with no detectable side-reaction products. Labeled receptors A and B were digested with Staphylococcus aureus V8 protease to yield smaller [3H]R5020-protein fragments derived from both. Molecular weight estimates (Mr = 9,500) and apparent isoelectric points indicate similarities of these regions of both A and B. The photoaffinity protocol described here thus provides a method for study of the hormone-binding domain of progesterone receptors and of receptor proteolysis in crude extracts.

Affinity Labels↗
PubMed1982

Photoaffinity labeling of insulin receptors in viable cultured human lymphocytes. Demonstration of receptor shedding and degradation.

A photosensitive derivative of radiolabeled insulin, SANAH-125I-insulin, was prepared by reacting N-succinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate (SANAH) with 125I-insulin. Cultured IM-9 cells were incubated with SANAH-125I-insulin at 16 degrees C in the dark. They were then washed, photolyzed, solubilized, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Under disulfide reducing conditions, a single specific band of Mr 125,000 was obtained. The characteristics of the labeling of this band with SANAH-125I-insulin (specificity, time course, concentration effect) were the same as that of 125I-insulin interaction with the IM-9 cells and the labeling process did not affect cell viability. The solubilized photolabeled insulin receptor fraction was enriched by first adsorbing to agarose-bound wheat germ agglutinin and the material eluted with N-acetyl-D-glucosamine was then analyzed by SDS-PAGE and autoradiography. Under nonreducing conditions, a major receptor band of Mr 320 K and a minor band of 280 K were obtained. Upon disulfide bond reduction with increasing concentrations of dithiothreitol, a major band of Mr 125 K and two minor bands of Mr 210 K and 94 K were seen. When cells photolabeled at 16 degrees C were further incubated at 37 degrees C, there was a time-dependent loss of intact receptors into the incubation buffer. In contrast, no similar shedding of labeled receptors was observed from isolated rat adipocytes. Following shedding, the labeled IM-9 insulin receptors rapidly disappeared from the incubation buffer (half-time approximately 1.5 h). These results demonstrate the feasibility of photoaffinity labeling, characterizing, and following the fate of insulin receptor in viable cells. Thus receptor photoaffinity labeling should provide a suitable approach for studies of the biologic fate of insulin receptors in cells that are targets for insulin action.

Autoradiography↗
PubMed1986

Photoaffinity labeling of the K562 cell membrane D-glucose transporter with cytochalasin B.

D-glucose carrier protein in K562 cell membrane was studied by photoaffinity labeling with cytochalasin B. The saturable cytochalasin B binding in purified K562 cell membranes was 90 pmol/mg and 200 pmol/mg protein in the presence of D-glucose and D-sorbitol, respectively. More than half of the total cytochalasin B binding could be depressed by D-glucose. The results of SDS-PAGE analysis of K562 cell membranes after photoaffinity labeling at 0.1 microM cytochalasin B showed that the main peak of covalently bound [3H]-cytochalasin B was in the Mr range of 46-65 KDa. The label found in the peak was reduced by more than 50% in the presence of 0.5 M D-glucose, the inhibition similar being to that obtained in the binding experiment. This polypeptide has a slightly higher molecular weight than that of the human erythrocyte cell membrane.

Affinity Labels↗
PubMed1995

Identification of a 23 kDa protein from maize photoaffinity-labelled with 5-azido-[7-3H]indol-3-ylacetic acid.

A 23 kDa protein (p23) was identified in microsomal extracts from maize coleoptiles by photoaffinity labelling with 5-azido-[7-3H]indol-3-ylacetic acid ([3H]N3IAA). Labelling of p23 was blocked by unlabelled IAA, N3IAA, indol-3-ylbutyric acid and indol-3-yl-lactate. In addition, labelling was efficiently decreased by tryptophan, as well as by the scavenger p-aminobenzoic acid. Labelling was, however, not affected by synthetic auxins such as 1-naphthylacetic acid or 2,4-dichlorophenoxyacetic acid. Competition data suggest that the label was probably bound via the indole ring, and hence labelling was not specific for auxins. The 23 kDa protein was solubilized from crude microsomes by extraction with Triton X-100 and purified to homogeneity by ion-exchange, size-exclusion and reversed-phase chromatography. After electroblotting, the amino acid sequences of the p23 N-terminus as well as the several tryptic peptides were obtained. Database comparisons revealed sequence identity with a maize manganese superoxide dismutase. We conclude that photoaffinity labelling of p23 was pseudo-affinity, and therefore the binding site for IAA is not specific.

4-Aminobenzoic Acid↗
PubMed2001

Application of photoaffinity labeling with [(3)H] all trans- and 9-cis-retinoic acids for characterization of cellular retinoic acid--binding proteins I and II.

Cellular retinoic acid-binding proteins (CRABPs) are carrier proteins thought to play a crucial role in the transport and metabolism of all-trans-retinoic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity-based binding assay involving competition between potential ligands of CRABP and [(3)H]atRA or [(3)H]-9-cis-RA for binding to the atRA-binding sites of CRABP I and II. Photoaffinity labeling of purified CRABPs with [(3)H]atRA was light- and concentration-dependent, saturable, and protected by several retinoids in a concentration-dependent manner, indicating that binding occurred in the CRABP atRA-binding site. Structure-function relationship studies demonstrated that oxidative changes to the atRA beta-ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs. These studies also identified two novel ligands for CRABPs: 5,6-epoxy-RA and retinoyl-beta-D-glucuronide (RAG). The labeling of both CRABPs with 9-cis-RA occurred with much lower affinity. Experimental evidence excluded nonspecific binding of RAG to CRABPs and UDP-glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an effective ligand of CRABPs. Therefore, photoaffinity labeling with [(3)H]atRA can be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) localized in the atRA-binding site of these proteins.

Alitretinoin↗
PubMed1987

Phylogenetic comparison of the photoaffinity-labeled benzodiazepine receptor subunits.

The late evolutionary appearance of the benzodiazepine receptor (BZR) allows an experimental approach for evaluation of the qualitative development of its subunits. Photoaffinity labeling of brain membranes with [3H]flunitrazepam followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography offers a suitable method for tracing the qualitative evolution of the BZR. A systematic comparison of the subunit patterns in fishes, amphibians, reptiles, birds, and mammals revealed that the subunit of 53K is phylogenetically the oldest photoaffinity labeled subunit; whereas it is the only band present in the lungfish and most amphibians, additional bands are apparent in higher tetrapods. In fishes, the evolution of the BZR subunits leads to the loss of the 53K subunit. KD values are discussed in relation to specific subunit patterns. Possible explanations for the observed variation of the subunits are discussed, with special emphasis placed on the possible evolution by gene duplication and subsequent divergence.

Amphibians↗