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PubMed1993

Photoaffinity labeling of the NMDA receptor.

The structure of NMDA receptors in situ has been probed with the novel photoaffinity ligand 125I-CGP 55802A. By covalently linking the radioactive high-affinity photolabel to NMDA receptors in bovine brain we have identified a protein of 175 kDa associated with the binding site for NMDA receptor agonists and competitive antagonists. Based on its molecular size the photolabeled protein is likely to correspond to the NR2A and/or NR2B subunit. The photoaffinity ligand will permit the assessment of regulatory changes in NMDA receptor subunit expression.

Affinity Labels↗
PubMed1987

Synthesis of spin-labeled photoaffinity derivatives of NAD+ and their interaction with lactate dehydrogenase.

The synthesis of NAD+ derivatives spin-labeled at either N6 or C8 of the adenine ring is described, in which the carboxamide function of the nicotinamide moiety is replaced by a diazirine ring. Irradiation of these compounds at 350 nm generates a carbene which will react with any functional group in its vicinity including hydrocarbons. Both NAD+ derivatives form tight ternary complexes with lactate dehydrogenase and were covalently incorporated into this enzyme. They may be employed for ESR studies when non-covalent interactions are too weak for motionally restricted species to be observed.

Animals↗
PubMed1984

Photoaffinity labeling of turkey erythrocyte beta-adrenergic receptors: degradation of the Mr = 49,000 protein explains apparent heterogeneity.

The potent photoaffinity probe [125I]p-azidobenzylcarazolol was used to identify beta-adrenergic receptors from turkey erythrocytes. Two peptides were specifically labeled with apparent Mr = 39,000 and 49,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The ratio of labeling of these peptides was found to be dependent on the method of tissue preparation. Thus, the Mr = 39,000/Mr = 49,000 ratio was 1:2 when labeling was done on intact cells, 1:1 for labeling of crude membranes, and 4:1 for purified membrane preparations. Moreover, incubation of intact cells or either membrane preparation at 37 degrees C led to a diminution of the Mr = 49,000 labeled peptide which was associated with a stoichiometric increase in the Mr = 39,000 form of the receptor. These results suggest that the turkey erythrocyte beta-adrenergic receptor is a protein of Mr = 49,000 and that the commonly observed Mr = 39,000 peptide is derived from this protein.

Affinity Labels↗
PubMed1984

Photoaffinity labeling of different benzodiazepine receptors at physiological temperature.

Irreversible labeling of benzodiazepine receptors in membranes from cerebellum or hippocampus was compared at 0 degrees C using [3H]flunitrazepam as a photoaffinity ligand. [3H]Flunitrazepam reproducibly and irreversibly labeled mainly one protein (P51) in cerebellum and at least two proteins (P51 and P55) in hippocampus at both temperatures. Differential inhibition at 37 degrees C of irreversible [3H]flunitrazepam binding to the individual proteins by several selective benzodiazepine receptor ligands supports the hypothesis that P51 and P55 are associated with different benzodiazepine receptors.

Affinity Labels↗
PubMed1986

Photoaffinity labelling of gizzard myosin with 3'-O-(4-benzoyl)-benzoic-adenosine 5'-triphosphate.

The reaction of a photoaffinity analog, 3'-O-(4-benzoyl)-benzoic-adenosine 5'-triphosphate (BZ2ATP) with gizzard myosin is described. The incorporation of BZ2ATP into myosin is both specific and stoichiometric. About 2.2 mol BZ2ATP are incorporated/mol myosin resulting in the significant loss of EDTA(K+) ATPase activity. The Mg2+ and actin-activated ATPase activities are slightly inhibited. Addition of ATP (millimolar) during the photolysis reaction significantly inhibits incorporation of BZ2ATP into myosin. Our data show that the label is mainly incorporated into the heavy chain of myosin with some label in the 20-kDa light chain. Limited proteolysis of radioactively labeled myosin subfragment 1 with trypsin reveals the presence of radioactivity mainly in the 50-kDa fragment and some in the 29-kDa and 25-kDa fragments. However, our data on the ATP-sensitive incorporation of BZ2ATP into the tryptic fragments suggest that the 50-kDa peptide, not the 29-kDa peptide, may be located at or around the active site.

Adenosine Triphosphatases↗
PubMed1991

Synthesis of 5-nitro-2-[N-3-(4-azidophenyl)-propylamino]-benzoic acid: photoaffinity labeling of human red blood cell ghosts with a 5-nitro-2-(3-phenylpropylamino)-benzoic acid analog.

A photoaffinity analog of the potent epithelial chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid has been synthesized and characterized. In the dark, this reagent, 5-nitro-2-[N-3-(4-azidophenyl)-propylamino]-benzoic acid, and the parent compound reversibly inhibited chloride efflux in human red blood cell ghosts. Irradiation of ghost membranes with 350 microM arylazide analog reduced the rate of chloride efflux to 33% of the control value. The photoinactivation process was not reversed by exhaustive washing of ghost membranes. Covalent incorporation of the photoaffinity reagent was supported by difference ultraviolet spectroscopy, which indicated the attachment of the substituted 2-amino-5-nitrobenzoic acid chromophore to ghost membranes. The novel photolabeling agent described here should be a useful structural probe for chloride channels in erythrocyte membranes and epithelial cells.

Affinity Labels↗
PubMed1985

Differential photoaffinity labelling of serotonin-S2 receptors and histamine-H1 receptors using 7-azidoketanserin.

7-Azidoketanserin, a potent photoaffinity probe for serotonin-S2 receptors was shown to irreversibly photoinactivate histamine-H1 receptors as well. The photolabelling of H1-receptors could be prevented by several selective histamine-H1 antagonists. In guinea pig cerebellum, a brain area that is highly enriched in H1-receptors, photolabelling could be directed exclusively to these receptors by adding a high concentration of pipamperone, which selectively blocks serotonin-S2 receptors. In rat pre-frontal cortex, a region that is enriched in serotonin-S2 receptors, pyrilamine was used to block H1-receptors, thereby directing the photolabelling exclusively to S2-receptors.

Affinity Labels↗
PubMed1989

Photoaffinity labeling of tubulin with (2-nitro-4-azidophenyl)deacetylcolchicine: direct evidence for two colchicine binding sites.

A new photoaffinity analogue of colchicine, (2-nitro-4-azidophenyl)deacetylcolchicine (NAPDAC), bound to two classes of sites on bovine renal tubulin and photolabeled both the alpha- and beta-subunits. The apparent Ki for the photoaffinity analogue was 1.40 +/- 0.17 microM (mean +/- SD, n = 3) as measured by competition with [3H] colchicine. Values of the apparent KdS for the two sites, as measured by the direct binding of the [3H]NAPDAC to tubulin, were 0.48 +/- 0.11 microM and 11.6 +/- 3.5 microM (mean +/- SD, n = 6), and the corresponding stoichiometries of binding of the two sites were 0.25 +/- 0.06 and 1.3 +/- 0.4 mol/mol of tubulin (mean +/- SD, n = 6). NAPDAC was a potent inhibitor of microtubule formation as detected by electron microscopy. When tubulin was photolabeled with NAPDAC at 25 degrees C, 15 +/- 3 mol % (mean +/- SD, n = 6) of the [3H]NAPDAC was covalently bound to the alpha-subunit, and 67 +/- 9 mol % (mean +/- SD, n = 6) was covalently bound to the beta-subunit. Since NAPDAC is a mixture of two interconvertible diastereomers, the photoincorporation of each was also examined. One diastereomer photolabeled both alpha- and beta-tubulin; however, the other did not significantly photolabel either subunit. Tubulin photolabeled with NAPDAC (1:1 mole ratio) exhibited a 23% decrease in colchicine binding. Preblocking and prephotolysis experiments with colchicine, NAPDAC, or ANPAH-CLC [Williams et al. (1985) J. Biol. Chem. 260, 13794-13802] provided evidence for conformational changes in tubulin upon colchicine binding. Peptide maps of [3H]NAPDAC-labeled alpha- and beta-tubulin, using Staphylococcus aureus V8 protease, demonstrated the presence of NAPDAC in one peptide of the alpha-subunit and in five peptides of the beta-subunit as detected by autoradiography. NAPDAC provides the first direct evidence for two colchicine binding sites on tubulin.

Affinity Labels↗
PubMed1981

Photoaffinity labeling of the cell surface adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum and its modification in down-regulated cells.

The cAMP cell surface receptor of Dictyostelium discoideum amoebae was identified by the use of the photoaffinity analogue 8-N3-[32P]cAMP. Labeling by intact cells of one component, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, could be specifically inhibited by the presence of nonradioactive cAMP. The component, P45 (apparent molecular weight of 45,000), was not identified on vegetative cells but was labeled with increasing intensity as cells differentiated and increased their levels of surface cAMP binding sites. Developmental mutants, starved under conditions where they do not express significant levels of cAMP binding sites, did not incorporate radioactivity into this protein. These mutants did label P45 when starved under differentiation-inducing conditions such that their levels of surface cAMP binding sites increased. P45 co-purified with the plasma membrane fraction isolated from cells to which 8-N3-[32p]cAMP had been covalently bound. Down-regulated amoebae, which displayed approximately 25% of the binding activity of untreated cells, did not label P45. These cells did, however, label a new component with an apparent molecular weight of 47,000 (P47).l The appearance of this component represented the only discernible difference in labeling profile under these conditions. As in the case of P45, radioactive incorporation into P47 did not occur if the photoactivation of 8-N3-[32P]cAMP was performed in the presence of nonradioactive cAMP.

Affinity Labels↗
PubMed1989

Photoaffinity labeling of terminal deoxynucleotidyl transferase. 1. Active site directed interactions with 8-azido-2'-deoxyadenosine 5'-triphosphate.

A photoaffinity analogue of dATP, 8-azido-2'-deoxyadenosine 5'-triphosphate (8-azido-dATP), was used to probe the nucleotide binding site of the non-template-directed DNA polymerase terminal deoxynucleotidyl transferase (EC 2.7.7.31). The Mg2+ form of 8-azido-dATP was shown to be an efficient enzyme substrate with a Km of 53 microM. Loss of enzyme activity occurred during UV photolysis only in the presence of 8-azido-dATP. At saturation (120 microM 8-azido-dATP), 54% of the protein molecules were modified as determined by inhibition of enzyme activity. Kinetic analysis of enzyme inhibition induced by photoincorporation of 8-azido-dATP indicated an apparent Kd of approximately 38 microM. Addition of 2 mM dATP to 120 microM 8-azido-dATP resulted in greater than 90% protection from photoinduced loss of enzyme activity. In contrast, no protection was observed with the addition of 2 mM dAMP. Enzyme inactivation was directly correlated with incorporation of radiolabeled 8-azido-dATP into the protein and UV-induced destruction of the azido group. Photoincorporation of 8-azido-dATP into terminal transferase was reduced by all purine and pyrimidine deoxynucleoside triphosphates of which dGTP was the most effective. The alpha and beta polypeptides of calf terminal transferase were specifically photolabeled by [gamma-32P]-8-azido-dATP, and both polypeptides were equally protected by all four deoxynucleoside triphosphates. This suggests that the nucleotide binding domain involves components from both polypeptides.

Adenosine Triphosphate↗
PubMed1990

Immunochemical detection of photoaffinity-labelled capsaicin-binding proteins from sensory neurons.

Capsaicin is a plant neurotoxin which depolarises a subset of mammalian sensory neurons. A photoaffinity probe (4-azidophenylpropionamide) with capsaicin-like agonist activity (EC50 5 microM) has been used to covalently label rat and chick sensory neurons in culture, as well as membrane preparations from both neurons and other tissues. Dorsal root ganglion cell specific capsaicin-binding proteins, including a major band of apparent molecular mass 58,000, have been identified by means of Western blotting, using a specific anti-capsaicin antiserum characterised by radioimmunoassay with a large range of capsaicin congeners. Using the same radioimmunoassay, no endogenous capsaicin-like immunoreactive material in normal or inflamed tissue has, however, been detected.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of human platelet and rabbit kidney alpha 2-adrenoceptors with [3H]SKF 102229.

A newly developed alpha 2-adrenergic photoaffinity ligand, 3-methyl-6-chloro-9-azido-1H-2,3,4,5-tetrahydro-3-benzazepine (SKF 102229), has been radiolabeled with tritium to a specific activity of approximately 80 Ci/mmol. Using membranes prepared from human platelets and from rabbit kidney, alpha 2-adrenoceptors have been covalently labeled following photolysis in the presence of [3H]SKF 102229. As determined by SDS-PAGE, the apparent molecular weight of alpha 2-adrenoceptors from both of these tissues was 64,000. The yield of covalent insertion of [3H]SKF 102229 into the alpha 2-adrenoceptor was very good. Thus, following photolysis up to 90% of the alpha 2-adrenoceptors could be irreversibly labeled with [3H]SKF 102229.

Affinity Labels↗
PubMed1985

Photoaffinity labeling of the DDT1 MF-2 cell alpha 1-adrenergic receptor.

In this study, we have used an alpha 1-adrenergic receptor photoaffinity ligand, 2-[4-(4-azido-3-iodo-benzoyl)-piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (125I-APD), to label covalently the alpha 1-adrenergic receptor in a smooth muscle cell line. Our results indicate that in the absence of light, (125I)APD binds reversibly to a site in the DDT1 MF-2 cell membranes having pharmacological characteristics of an alpha 1-adrenergic receptor. Following incorporation of (125I)APD into partially purified membranes a single labeled band of protein with a Mr of 81 000 was visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incorporation of (125I)-APD into this band was affected by adrenergic agonists and antagonists in a manner consistent with an alpha 1-adrenergic interaction. Prazosin (alpha 1-selective) blocked incorporation of the label into the Mr = 81 000 protein while yohimbine (alpha 2-selective) did not. Of the adrenergic agonists, (-)-epinephrine and (-)-norepinephrine but not (-)-isoproterenol blocked labeling of the Mr = 81 000 protein. We conclude that the ligand binding site of the DDT1 MF-2 cell alpha 1-adrenergic receptor resides in a Mr = 81 000 protein.

Adrenergic alpha-Agonists↗
PubMed1992

Identification of actin and HSP 70 as cyclosporin A binding proteins by photoaffinity labeling and fluorescence displacement assays.

A novel family of cyclosporin A (CsA) binding proteins was identified by using the biologically active, radioiodinated photoaffinity probe [D-Lys-N epsilon-(4-azido-3-[125I]iodophenyl)propionyl)]8-CsA. In addition to cyclophilin, proteins with molecular masses of 43 kDa and approximately 50-55 kDa were labeled in Jurkat extracts and bovine calf thymus. Sequence analysis of the 43-kDa protein purified from calf thymus and subsequent Western analysis of CsA affinity-purified material from Jurkat extracts identified the 43-kDa component as actin. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA, a fluorescent analogue of CsA, was prepared and used to measure the binding constants of cyclosporin derivatives to actin by means of a new fluorescence displacement assay. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA and [N delta-t-butoxycarbonyl diaminobutyryl)]8-CsA bind to bovine actin at physiologically relevant concentrations, with dissociation constants of 60 +/- 33 and 570 +/- 380 nM, respectively. Because the ATPase fragment of heat shock cognate 70 (HSC 70) is structurally related to actin, the yeast homologue SSA1 was tested and found to be radiolabeled by the cyclosporin A photoaffinity reagent. The binding constant for [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA to SSA1 was determined and is 53 +/- 48 nM. These results indicate that actin and the 70-kDa heat shock protein family contain a structurally related domain for binding of cyclosporin A-related peptides.

Actins↗
PubMed1985

Photoaffinity labeling of an antimycin-binding site in Rhodopseudomonas sphaeroides.

Tritium-labeled 3-azidosalicyl-N-(n-octadecyl)amide was synthesized and used as a photoaffinity probe for the antimycin-binding site in both purified ubiquinone-cytochrome b-c1 oxidoreductase and chromatophore vesicles from the photosynthetic bacterium Rhodopseudomonas sphaeroides. In both systems, a prominently labeled protein had a molecular weight of 11,000. Binding to this protein was inhibited by preincubation of the reaction mixture with antimycin prior to addition of the radioactive analog and subsequent irradiation. The antimycin analog, 3-azidosalicyl-N-(n-octadecyl)amide, inhibited succinate-cytochrome c reductase activity in chromatophore vesicles by 50% at a concentration of 150 nmols/mg of protein.

Affinity Labels↗
PubMed1989

Two different regions of P-glycoprotein [corrected] are photoaffinity-labeled by azidopine.

Cells that express P-glycoprotein are resistant to many unrelated anticancer drugs. All evidence suggests that P-glycoprotein is a plasma membrane protein that confers multidrug resistance by actively transporting these cytotoxic drugs out of cells. The objective of our work is to locate drug binding sites on P-glycoprotein. Azidopine is a photoaffinity drug analog that specifically labels P-glycoprotein. To determine the region of P-glycoprotein that binds azidopine, we labeled P-glycoprotein with azidopine and digested the labeled protein into fragments. We then identified the labeled fragments with specific antibodies. We have determined that azidopine labels two different regions of P-glycoprotein: one region is in the amino half of P-glycoprotein, and the other is in the carboxyl half of the protein. Our results suggest that P-glycoprotein contains either two binding sites for azidopine or a single site formed by the two homologous halves of the protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Design, synthesis and utility of novel benzophenone-containing calcitonin analogs for photoaffinity labeling the calcitonin receptor.

Calcitonin (CT) is a 32-amino-acid calciotropic peptide hormone which acts on target cells via a G protein-coupled seven-transmembrane receptor (CTR). In this study, we report the design, synthesis and characterization of four potent bioactive and photoreactive CT analogs, each of which contains a single benzophenone moiety inserted at different and discrete locations within the CT molecule. Replacement of all Lys residues in salmon CT (sCT) with Arg, followed by replacement of hydrophobic residues with a Lys(epsilon-p-benzoylbenzoyl) residue [Lys(epsilon-pBz2)] was found to preserve high biological activity. We substituted Val8, Leu16 and Leu19 by Lys(epsilon-pBz2), and acylated the N-terminus by a pBz2 moiety, thus distributing the photoaffinity moiety in the different analogs across a large portion of the CT sequence. With both transfected and endogenous CTRs from several species, all four benzophenone-containing analogs were shown to be virtually indistinguishable from the parent sCT analog in both receptor binding properties and stimulation of cAMP accumulation. Upon photolysis, in the presence of CTR, the radioiodinated photoreactive CT analog ([Arg11,18,Lys19(epsilon-pBz2)]sCT (K19)) covalently labels a membrane component of approximately 70 kDa. Receptor cross-linking is inhibited specifically in the presence of excess sCT. We also examined the interaction of these CT analogs with a hemagglutinin (HA) epitope-tagged CTR. The HA-CTR displayed CT binding and CT-dependent cAMP stimulation identical with native CTR. Both K19 and another bioactive analog (-Arg11,18, Lys8(epsilon-pBz2)]sCT (K8)) specifically photoaffinity cross-link to the HA-CTR. These benzophenone-containing CT analogs should facilitate studies of hormone-receptor interactions and allow the direct identification of a CT binding domain(s) within the receptor by the analysis of photochemically cross-linked conjugates.

Amino Acid Sequence↗
PubMed1988

Photoaffinity labeling of opioid delta receptors with an iodinated azido-ligand: [125I][D-Thr2,pN3Phe4,Leu5]enkephalyl-Thr6.

The photoaffinity ligand of the delta opioid receptor Tyr-D-Thr-Gly-pN3Phe-Leu-Thr (azido-DTLET) was iodinated and purified by high performance liquid chromatography. Monoiodo-azido-DTLET displayed a high affinity (KD = 15 nM) and is selective (Kl mu/Kl delta = 9.8) for rat brain delta opioid receptors (for comparison, the corresponding values for tritiated azido-DTLET are KD = 1.66 nM and Kl mu/Kl delta = 27). On rat brain sections, the anatomical distribution of [125I]azido-DTLET binding sites revealed by autoradiography corresponds to that of delta receptors. On rat brain membrane homogenates and NG108-15 hybrid cells, UV irradiation of the receptor-ligand complex results in the irreversible binding to membrane proteins of 14% of the bound radioactivity Gel electrophoresis of [125I]azido-DTLET-labeled proteins followed by autoradiography shows a different pattern in rat brain and NG108-15 cells. In rat brain, labeling of two of these proteins, with molecular weights of 44,000 and 34,000, was inhibited by 30 nmol/liter of nonradioactive DTLET, a delta-selective ligand but not by the same concentration of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a mu-selective ligand. In NG108-15 cells, this 44-kDa protein was not visualized; the main band was at 33 kDa and disappeared in the presence of levorphanol.

Affinity Labels↗