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At least 19 recordsLinked to original sources
PubMed1973

The mechanism of photoaffinity labeling.

Photoaffinity labeling is a recently introduced method for covalently binding chemical tags to the active sites of protein molecules, which is potentially capable of very great specificities of labeling. A labeling reagent is used that is converted by photolysis to an extremely reactive intermediate. According to the expected mechanism, the reagent molecules that are specifically and reversibly bound to the active site at the instant of photolysis react irreversibly in the site before they can dissociate from the site. In two such reagent-protein systems studied in this paper, however, it is shown that, while by the usual criteria photoaffinity labeling appears to have occurred, the expected mechanism in fact does not hold. This was discovered in experiments with scavengers present in the mixtures that were photolyzed. The general properties of, and criteria for, photoaffinity labeling reactions are discussed in the light of these findings.

Acetylcholinesterase↗
PubMed1986

Identification of the D2-dopamine receptor binding subunit in several mammalian tissues and species by photoaffinity labeling.

Photoaffinity labeling of the D2-dopamine receptor in plasma membrane preparations of various tissues from several mammalian species was performed using the recently developed D2-dopaminergic antagonist probe [125I]N-(p-azidophenethyl)spiperone ([125I]N3-NAPS). In tissues containing D2-receptors such as the corpus striatum from rat, dog, calf, hamster, guinea pig, and rabbit as well as the anterior pituitary of rat, bovine, and hamster, the probe covalently labels a peptide of Mr = 94,000. Specificity of the labeling is typically D2-dopaminergic in character. The covalent labeling is blocked by (+)-butaclamol but not by the inactive (-)isomer. Agonists block incorporation with the order of potency: N-n-propylnorapomorphine greater than apomorphine greater than dopamine. The D2-selective antagonist spiperone blocks labeling of the Mr = 94,000 peptide whereas the D1-selective antagonist SCH-23390 is ineffective. Thus, these results indicate that the ligand binding subunit of the D2-dopamine receptor resides on a Mr = 94,000 peptide in these various tissues from several species. Under conditions where proteolysis is not stringently controlled, peptides of lower Mr (32-38,000) are labeled at the expense of the Mr = 94,000 peptide. The most efficient protease inhibitor tested in these systems was EDTA, suggesting that the generation of these lower Mr receptor fragments might be the result of a metal-dependent proteolysis in the membrane preparations. In the rat neurointermediate lobe, a tissue containing D2-receptors, [125I]N3-NAPS specifically labels a major peptide of Mr approximately equal to 120,000 in addition to the Mr = 94,000 peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1984

Ethidium binding sites on plasmid DNA determined by photoaffinity labeling.

Photoaffinity labeling of pBR322 with ethidium monoazide (8-azido-3-amino-5-ethyl-6-phenylphenanthridinium chloride) was used to provide evidence for the sequence specificity of ethidium binding to native DNA. DNA-drug interactions were examined at concentrations of eight covalently bound ethidium drugs per molecule of pBR322 (4363 base pairs). Restriction enzyme cutting was blocked by the covalent binding of a drug molecule at (or near) the enzyme recognition sequence. This phenomenon was observed with all restriction enzymes tested and was not limited to specific regions of the pBR322 molecule. Double-digestion experiments indicated that a drug molecule may bind 2 to 3 base pairs outside the recognition sequence and still block restriction enzyme digestion. Intact plasmid was treated with [3H]ethidium monoazide and digested with restriction enzymes. The amount of covalently-linked ethidium analog was quantitated for different restriction fragments and the G-C content of each fragment was determined from the DNA sequence. In approximately half of the fragments the drug appeared to preferentially bind at a G-C base pair. However, no preference for specific sequences such as 5'-C-G-3' was detected, as had been suggested by previous modeling studies with ethidium bromide. The other fragments were located in specific map regions of the plasmid and did not bind drug with a strict dependence on GC content suggesting that binding specificity may depend on more than one structural feature of the DNA.

Affinity Labels↗
PubMed1979

Bile salt binding to serum components. Taurocholate incorporation into high-density lipoprotein revealed by photoaffinity labelling.

1. Photoaffinity labelling of human serum albumin with the sodium salts of (3 beta-azido-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino[2(-3)H (N)]ethanesulfonic acid, (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino[2(-3)H (N)]ethanesulfonic acid and (11 zeta-azido-12-oxo-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-amino[2(-3)H (N)]ethanesulfonic acid resulted, in each case, in a considerable covalent incorporation of radioactivity into the protein. 2. Photoaffinity labelling of whole serum, obtained from fasting test persons, revealed with all three photolabile derivatives of taurocholate at the physiological concentration of 2.1 microM the incorporation of radioactivity not only into albumin but also into high-density lipoprotein, as demonstrated by density gradient centrifugation and by immunological characterization. 3. The bulk of radioactivity incorporated into high-density lipoprotein by photoaffinity labelling of whole serum was found to have been associated with the lipids. Only 10-20% of the label was covalently bound to apolipoproteins, predominantly to the apolipoproteins A-I and A-II. 4. The interaction of taurocholate with high-density lipoprotein has been confirmed by density gradient centrifugation using 14C-labelled taurcholate. It is assumed that the interaction of taurocholate with high-density lipoprotein is physiologically of significance.

Adult↗
PubMed1991

Detection of cannabinoid receptors by photoaffinity labelling.

A novel [125I]-labelled photoaffinity ligand designed to detect cannabinoid binding sites has been used in mouse brain preparations and in cultured S49 mouse lymphoma cells. The ligand, 2-iodo-5'-azido-delta 8-THC, shows a high affinity for sites in both brain (Kd = 5.60 pM) and whole cell (Kd = 9.38 pM) systems. Photolabelling studies with brain samples revealed the existence of four ligand-protein adducts, of estimated molecular weights 85.5, 62.1, 30.0 and 25.5 kDa, that were diminished by prior exposure to 8 microM THC. A similar study with S49 cells gave adducts with apparent molecular weights of 62.1, 34.4, 16.9 and 13.5 kDa. The ligand produces a typical cannabinoid cataleptic response in mice suggesting that possibly one or more of the binding sites may be involved in some of the receptor mediated actions of THC.

Affinity Labels↗
PubMed1988

Deletion of cAMP-binding site B in the regulatory subunit of cAMP-dependent protein kinase alters the photoaffinity labeling of site A.

Photoaffinity labeling with 8-azidoadenosine 3':5'-monophosphate is a highly selective method for probing the cAMP-binding sites of the regulatory subunits of cAMP-dependent protein kinase and for identifying specific residues that are in close proximity to the cAMP-binding sites. The cAMP-binding site of a mutant RI-subunit has been characterized here and contrasted to the native RI-subunit. This mutant RI-subunit was generated by oligonucleotide-directed muta-genesis and lacks the entire second cAMP-binding domain which includes both of the residues, Trp260 and Tyr371, that are photolabeled in the native RI-subunit. The mutant RI-subunit, nevertheless, is photoaffinity-labeled with high efficiency, and the residue covalently modified was identified as Tyr244. The position of Tyr244 based on a computer graphic model of cAMP-binding site A is proposed and correlated with the presumed locations of Tyr371 and Trp260 in the native R-subunit. Photoaffinity labeling also can be used to detect functional cAMP-binding sites following electrophoretic transfer of the denatured protein to nitrocellulose. Labeling of the immobilized protein on nitrocellulose required a functional cAMP-binding site A that can be photoaffinity-labeled in solution based on the following criteria. 1) The type I R-subunit is photolabeled, whereas the type II R-subunit is not. A primary feature which distinguishes these two R-subunits is that the RI-subunit is photolabeled at both sites A and B, whereas covalent modification of the RII-subunit occurs only at site B. 2) The truncated mutant of the RI-subunit which lacks the entire second cAMP-binding domain can be photolabeled on nitrocellulose. 3) A mutant RI-subunit which can no longer be photolabeled in site B is still photolabeled on nitrocellulose. 4) A mutation which abolished cAMP binding to site A also abolished photoaffinity labeling after transfer to nitrocellulose.

Affinity Labels↗
PubMed1981

Early steps in the path of nascent ribonucleic acid across the surface of ribonucleic acid polymerase, determined by photoaffinity labeling.

The photoaffinity probes beta-(4-azidophenyl) adenosine 5'-diphosphate (N3PhppA) and beta-(4-azidophenyl) adenylyl-(3'--5')-uridine 5'-diphosphate (N3PhppApU) were used to determine the RNA polymerase subunit contacts made by the 5' ends of three nascent RNA chains. Ternary enzyme-poly[d(A-T)].oligonucleotide complexes were prepared in which the nascent oligonucleotide contained a photoaffinity label at the 5' end and a 32P radiolabel only at the 3' end. The length of the RNA was fixed at two, three, or four nucleotides. Photolysis of the ternary complexes was followed by dissociation, polyacrylamide gel electrophoresis, autoradiography, and scintillation counting. With a dinucleotide probe, the enzyme subunits labeled were beta' (71%) and sigma (21%). Photolysis of the ternary complex containing trinucleotide RNA also resulted in labeling of the beta' (64%) and sigma (35%) subunits. With a tetranucleotide, the beta' subunit was very heavily labeled (88%), and a small amount of labeling of the beta (7%) and sigma (4%) subunits was observed. The alpha subunit was not labeled with any of the probes. These results imply that a conformational change, possibly involving dissociation of the sigma subunit, occurs in the enzyme as the ribonucleotide is elongated from a tri- to a tetranucleotide.

Adenosine Diphosphate↗
PubMed1987

Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label.

An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin (IAPS-forskolin), has been synthesized, purified, and characterized. The I50 for inhibition of 3-O-methylglucose transport in red blood cells by IAPS-forskolin was found to be 0.05 microM. The carrier free radioiodinated label is a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes (ghosts) and purified glucose transporter preparations with 1-2 nM [125I]IAPS-forskolin and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed specific derivatization of a broad band with an apparent molecular mass of 40-70 kDa. Photoincorporation into erythrocyte membranes using 2 nM [125I]IAPS-forskolin was protected with D-glucose (I50 400 mM), cytochalasin B (I50 0.5 microM), and forskolin (I50 10 microM). No protection was observed with L-glucose (600 mM). Endo-beta-galactosidase digestion of [125I] IAPS-forskolin-labeled ghosts and purified transporter resulted in a dramatic sharpening of the specifically radiolabeled transporter to 40 kDa. Trypsinization of [125I]IAPS-forskolin-labeled ghosts and purified transporter reduced the specifically radiolabeled transporter to a sharp peak at 18 kDa. [125I]IAPS-forskolin will be a useful tool to study the structural aspects of the glucose transporter.

3-O-Methylglucose↗
PubMed1990

Detecting precatalytic conformational changes in F1-ATPase with 4-benzoyl(benzoyl)-1-amidofluorescein, a novel fluorescent nucleotide site-specific photoaffinity label.

A novel photoaffinity label for studies with the F1-ATPase has been synthesized and found to be an effective reporter of subunit conformational changes that occur in this enzyme upon multiple nucleotide-binding site occupancy. The new probe, 4-benzoyl(benzoyl)-1-amidofluorescein (BzAF), which possesses structural similarity to purine nucleotides, exhibits bifunctional characteristics that enable it to bind covalently to the exchangeable nucleotide sites on beef heart F1 (via photoactivation of the benzophenone moiety) and, once covalently linked, emit environmentally sensitive fluorescence (via selective excitation of the fluorescein moiety). BzAF binds competitively with ATP in the absence of illumination, with a KI of 50 microM. Under actinic irradiation necessary for generating the covalently reacting diradical triplet state of benzophenone, BzAF behaves as a nucleotide site-directed photoaffinity label of exchangeable (catalytic) sites, and the resulting photoinhibition of ATPase activity displays pseudo first-order rate-saturation kinetics that support formation of a dissociable BzAF.F1 complex (k-1/k1 = 58 microM) prior to covalent binding. The BzAF-induced photoinactivation is protectable with native nucleotide ligand (e.g. MgADP, Kprotect = 0.4 mM). Added corroboration of a catalytic cooperativity mechanism for F1 was obtained by finding a molar stoichiometric ratio [( 3H]BzAF:F1) of 1 required for complete inhibition of ATPase activity. Steady-state fluorescence studies with a unisite-labeled BzAF.F1 complex (a catalytically inactive species on which at least one exchangeable nucleotide-binding site remains unoccupied) display a saturable fluorescence quenching of the bound fluorescein upon titration with MgADP, but no change with MgAMP. These data imply that the filling of more than one of the catalytic binding sites/mol of F1 with nucleotide signals a precatalytic conformational adjustment that is transmitted between catalytic sites and across the beta-alpha-beta domain of the enzyme's subunit structure.

Affinity Labels↗
PubMed1987

Photoaffinity labeling of thyroid hormone receptors.

Photoaffinity label probes of iodothyronines can interact with nuclear receptors in intact cells and in solubilized receptor preparations. These probes have certain advantages over a chemical affinity label in analyzing receptor structure. First, a photoaffinity label probe covalently cross-links only after photoactivation. Therefore, it is possible to demonstrate with appropriate competitive inhibition studies that the photoaffinity label probe associates with the receptor in question. Secondly, since cross-linking only occurs after photolysis, it is possible to adjust the concentration of the photoaffinity label to maximize association with "specific" binding sites relative to "non-specific" associations prior to covalent linkage by photoactivation. The different [125I]iodothyronine-PAL analogues may be useful as probes of the thyroid hormone receptor binding domain since PAL compounds with different affinities for receptor may photocouple to different receptor residues within or proximate to the hormone binding region. These probes may also be useful as an adjunct to receptor purification and in probing the organization of the receptor in chromatin. Lastly, they may provide insights into possible alterations of receptor structure in patients with partial end organ resistance to thyroid hormone (Refetoff et al., 1967; Eil et al., 1982).

Affinity Labels↗
PubMed1983

[1,6-alpha-aminosuberic acid, 3-(p-azidophenylalanine), 8-arginine] vasopressin: a new photoaffinity label for hydroosmotic hormone receptors. Characterization of the ligand and irreversible stimulation of hydroosmotic water flow in toad bladder by photoaffinity labeling.

The photoreactive analog of vasopressin [1,6-alpha-aminosuberic acid, 3-(p-azidophenylalanine), 8-arginine] vasopressin [( Asu1,6, Phe (p-N3)3]AVP) has been synthesized. This analog retains a high binding affinity for the vasopressin receptor in plasma membranes from bovine kidney inner medulla (apparent dissociation constant, KD = 8.5 X 10(-9) M). [Asu1,6, Phe (p-N3)3] AVP was found to be biologically active in triggering the characteristic increase in toad bladder permeability to water. Photolysis of the analog in the presence of the toad bladder results in a hydroosmotic response which persists, in spite of repeated washings, for more than 18 h. The irreversible stimulation of the bladder is inhibited when photolysis is carried out in the presence of vasopressin. Our findings indicate that with photoactivation [Asu1,6, Phe(p-N3)3]AVP binds covalently to hormonal receptors and forms an active hormone-receptor complex. This analog, therefore, is a suitable tool for studies of hydroosmotic receptor function and for receptor isolation.

Affinity Labels↗
PubMed1991

Azido derivative of tricarboxylic acid for photoaffinity labeling.

A new photoaffinity probe, 5-(1-hydroxy-4-azidophenylazo)-1,2,3-benzenetricarboxylic acid, was synthesized and characterized. This reagent can be potentially used in photoaffinity labeling of the mitochondrial tricarboxylate carrier, as well as of enzymes interacting with tricarboxylic acids. Inhibition and labeling of the mitochondrial tricarboxylate carrier is presented.

Affinity Labels↗
PubMed1988

Membrane penetration of Sendai virus glycoproteins during the early stages of fusion with liposomes as determined by hydrophobic photoaffinity labeling.

The hydrophobic photoaffinity label 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine was used to label Sendai virus proteins during fusion with cardiolipin and phosphatidylserine liposomes. Preferential labeling of the viral fusion protein during the initial stages of fusion demonstrated that this protein interacts with the hydrophobic core of the target membrane as an initiating event of virus-liposome fusion. Labeling showed time, temperature, and pH dependence consistent with earlier fluorescent measurements of fusion kinetics. The present method provides conclusive evidence supporting the hypothesis that hydrophobic interaction of the fusion protein with the target bilayer is an initial event in the fusion mechanism of viral membranes.

Affinity Labels↗
PubMed1989

Identification of L-glutamate binding sites in chick brain by photoaffinity labeling.

The photoaffinity cross-linker, N-hydroxysuccinimidyl-4-azido benzoate ester, was used to attach L-[3H]glutamate irreversibly to chick brain membranes. Electrophoretic analysis with sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed a major radioactive protein band with an apparent molecular weight of 45,600 +/- 300 Da. A second band with a smaller amount of radioactivity and with an apparent molecular weight of 28,300 +/- 500 Da was also detected. Photolabeling was inhibited by quisqualic acid.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of the Ah receptor.

A series of halodibenzo-p-dioxins with the photolabile aryl azide functional group were synthesized and screened as potential photoaffinity labels for the Ah receptor, and 2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin was selected for radiosynthesis with 125I (specific activity 2176 Ci/mmol, equilibrium dissociation constant, KD = 0.76 nM). Following incubation of this 125I-labeled photoaffinity ligand with the protamine sulfate-precipitated fraction of C57BL/6J mouse liver cytosol, and irradiation with long wavelength ultraviolet light, the radiolabeled macromolecules were precipitated with acetone and analyzed by denaturing gel electrophoresis and autoradiography. Among the labeled products, two peptides with apparent molecular masses of 95,000 and 70,000 daltons had the following properties: 1) they were selectively labeled at low ligand concentrations; 2) they were labeled in approximately a 1:1 ratio; 3) co-incubation with receptor agonists inhibited the photoaffinity labeling of both peptides to a similar extent, and structure activity relationship for inhibition of labeling by these agonists corresponded to that for their binding affinity to the Ah receptor; 4) upon nondenaturing chromatographic separation of photoaffinity labeled cytosol on high performance liquid chromatography size exclusion and anion exchange columns, the 95- and 70-kDa peptides coelute; 5) the migration of these peptides upon denaturing electrophoresis is the same in the presence or absence of a thiol reducing agent; and 6) proteolysis of the 95- and 70-kDa peptides produces a similar pattern of cleavage peptides. The simplest structure of the Ah receptor in mouse liver cytosol, appears to be a dimer composed of two noncovalently linked subunits of apparent molecular masses of 95 and 70 kDa, which have homologous structure and similar ligand binding sites, but other possibilities are discussed.

Affinity Labels↗
PubMed1990

Photoaffinity labeling of RNA polymerase III transcription complexes by nascent RNA.

The proteins contacting nascent RNA transcripts in RNA polymerase III transcription complexes have been examined using photoaffinity labeling techniques. The photoaffinity analog 4-S-UTP was incorporated along with [alpha-32P]CTP into VAI transcripts, using a phosphocellulose fractionated HeLa S-100 extract and DNA containing the adenovirus VAI gene. The photoreactive nascent RNA was cross-linked to proximal proteins in the transcription complex. The photoaffinity labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by autoradiography. The specific photoaffinity labeling of RNA polymerase III was dependent on 4-S-UTP and on DNA containing a class III promoter. Photoaffinity labeling was inhibited by 200 micrograms/ml alpha-amanitin. Proteins of 140, 160, 270, and 310 kDa were labeled. These photoaffinity labeled proteins were shown to be stably associated with the DNA template by gel exclusion chromatography. The 160-kDa protein was cross-linked to RNAs approximately 14-18 nucleotides in length, whereas the greater than 250-kDa proteins were cross-linked to RNAs 18-30 nucleotides in length. The 140- and 160-kDa proteins correspond in molecular mass to the two large subunits of RNA polymerase III. The molecular masses of the 270- and 310-kDa proteins, and the length of the RNA cross-linked to them, suggest that these proteins are components of transcription factor (TF) IIIC. These results indicate that the nascent transcript contacts the two largest subunits of RNA polymerase III until the transcription complex reaches the TFIIIC binding site, at which point the nascent transcript contacts TFIIIC.

Adenoviridae↗
PubMed1988

Probing the peptide binding site of the cAMP-dependent protein kinase by using a peptide-based photoaffinity label.

A peptide-based photoaffinity label for the catalytic subunit of the cAMP-dependent protein kinase was prepared from the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)]. By using solid-phase peptide synthesis methodology, DL-Phe(pBz) was incorporated into the cAMP-dependent protein kinase substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in place of the phosphorylatable serine. The diastereomeric peptides were separated by reverse-phase HPLC. The peptide substrate analog containing L-Phe(pBz) had a Ki of approximately 110 microM at pH 7.5. When photolyzed at 350 nm in the presence of the enzyme, this peptide caused time- and concentration-dependent inactivation. Radioactive acetylated L-Phe(pBz) peptide was used to establish the binding stoichiometry of peptide to enzyme; these results, together with protection experiments, showed the photoaffinity labeling to be specific (approximately 1:1). To identify the residues that were modified on the catalytic subunit, the photoinactivated enzyme was cleaved with CNBr and V8 protease (Staphylococcus aureus). The resulting peptide fragments were purified by HPLC and were sequenced; these experiments identified the modified residues as Gly-125 and Met-127. This region of the cAMP-dependent protein kinase catalytic subunit contains many residues that are conserved in serine- and tyrosine-protein kinases.

Affinity Labels↗