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 541 records · Page 30Linked to original sources
PubMed1985

The subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ.

Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study (Branca, A. A., Sluss, P. M., Smith, A. A., and Reichert, L. E., Jr. (1985) J. Biol. Chem. 260, 9988-9993) in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84-kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs. The appearance of all bands, however, was inhibited by the inclusion of unlabeled hFSH in the initial binding incubation mixtures. The results of this study indicate that the calf testis FSH receptor has a multimeric structure containing at least one 48-kDa subunit and suggest the presence of other nonidentical receptor subunit proteins.

Acylation↗
PubMed1995

Synthesis of 5-IASA-UDP-GlcNAc and its use for the photoaffinity labeling of a novel UDP-GlcNAc pyrophosphorylase.

Photoreactivable 5-[3-(p-iodoazidosalicylamide)allyl]-UDP-GlcNAc (5-IASA-UDP-GlcNAc) was synthesized by a four-step procedure and used for photoaffinity labeling of UDP-GlcNAc-dependent enzymes. Upon iodination with 125I, the compound was successfully applied to probe a purified UDP-GlcNAc pyrophosphorylase from pig liver. The enzyme was photoinactivated by the probe in the concentration-dependent manner, and was protected by UDP-GlcNAc and, to a lesser extent, by UTP and UDP-GlcCOOH.

Affinity Labels↗
PubMed1988

Photoaffinity labeling of the oxysterol receptor.

A cytosolic receptor protein for oxygenated sterols, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol biosynthesis, can be labeled covalently by photoactivation of 7,7'-azo-[5,6-3H]cholestane-3 beta,25-diol. Other compounds tested for their potential as photoaffinity reagents were: 25-hydroxycholesta-4,6-dien-3-one, 3 beta,25-dihydroxycholest-5-en-7-one, and 3 beta-hydroxycholesta-8(14),9(11)-dien-15-one. These sterols did not bind to the receptor with adequate affinity, were not readily photolyzed, or did not react covalently with the receptor during photolysis. The successful photoaffinity label, 7,7'-azocholestane-3 beta,25-diol, binds to the receptor with high affinity (Kd = 9.1 nM). After activation of the partially purified oxysterol-receptor complex with UV light (greater than 300 nm), several covalently labeled proteins were found upon sodium dodecyl sulfate-gel electrophoresis. Labeling of one protein, Mr approximately 98,000, was much reduced when the binding reaction was carried out in the presence of an excess of unlabeled oxysterol. Under the reaction conditions investigated so far, approximately 1% of the specifically bound sterol was covalently linked after photolysis. These results are consistent with previous information suggesting that the Mr of the receptor subunit is approximately 97,000. The covalent labeling of the receptor reported herein should facilitate its further purification and characterization.

Affinity Labels↗
PubMed1986

Exofacial photoaffinity labelling of the human erythrocyte sugar transporter.

The 4-azidosalicylate derivative of 1,3-bis(D-mannos-4'-yloxy)-2-[2-3H]propylamine (ASA-[2-3H]BMPA) has been tested as a photoaffinity label for the sugar transporter in human erythrocytes. When photolysed in the presence of intact erythrocytes, ASA-[2-3H]BMPA covalently binds to the exofacial surface of the transporter. This labelled protein appears as a broad band in the 4.5 region in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The peak of radiolabel incorporation gives an apparent Mr of approx. 50 000 on 5-20% acrylamide gels. The binding is 80% inhibitable by 320 mM 4,6-O-ethylidene-D-glucose, by 320 mM D-glucose and by 50 microM cytochalasin B. Photoirradiation of a saturating concentration of ASA-BMPA in the presence of erythrocytes results in a 25-30% loss of D-galactose transport activity. From transport inactivation data and estimations of the amount of ASA-[2-3H]BMPA binding to the transporter it is calculated that there are approx. 220 000 exofacial hexose-transport binding sites per erythrocyte. The labelling of the transporter has been carried out using freshly drawn blood and 4-weeks-old transfusion blood. No change in the binding profile on SDS-polyacrylamide gel electrophoresis was observed. Proteolytic digestion of the ASA-[2-3H]BMPA-labelled transporter with either trypsin or alpha-chymotrypsin results in the appearance of a labelled 19 kDa fragment on SDS-polyacrylamide gel electrophoresis.

Affinity Labels↗
PubMed1985

Facilitated glucose transporter of human erythrocyte: proteolytic mapping of the [3H]cytochalasin B photoaffinity-labeled transporter polypeptide.

The human erythrocyte D-glucose transporter is an integral membrane glycoprotein with an heterogeneous molecular mass spanning a range 45-70 kDa. The protein structure of the transporter was investigated by photoaffinity labeling with [3H]cytochalasin B and fractionating the labeled transporter according to molecular mass by preparative SDS-polyacrylamide gel electrophoresis. Each fraction was digested with either papain or S. aureus V8 proteinase, and the labeled proteolytically derived peptide fragments were compared by SDS polyacrylamide gel electrophoresis. Papain digestion yielded two major peptide fragments, of approx. molecular mass 39 +/- 2 and 22 +/- 2 kDa; treatment with V8 proteinase resulted in two fragments, with mass of 24 +/- 2 and 15 +/- 2. Proteolysis of each transporter fraction produced the same pattern of labeled peptide fragments, irrespective of the molecular mass of the original fractions. The binding characteristics of [3H]cytochalasin-B-labeled transporter to Ricinis communis agglutinin lectin was examined for each transporter molecular mass fraction. It was found that higher-molecular-mass fractions of intact transporter had a 2-fold greater affinity for the lectin than lower-molecular-mass fractions (i.e., 67 kDa greater than 45 kDa fraction). However, proteolytically derived labeled peptide fragments from each fraction had minimal affinity for the lectin. These results suggest that the labeled peptide fragments have been separated from the glycosylated regions of the parent transporter protein. The present findings indicate that, although transporter proteins have an apparently heterogeneous molecular mass, some regions of the protein share a common peptide. Furthermore, the glycosylated regions appear to be located some distance from the [3H]cytochalasin-B-labeled site(s).

Affinity Labels↗
PubMed1979

Yeast phenylalanyl-tRNA synthetase. Affinity and photoaffinity labelling of the stereospecific binding sites.

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl-tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The RNAPhe was cross-linked to the enzyme by non-specific ultraviolet irradiation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4-thiouridine (S4U) residues introduced in the tRNA molecule, or by Schiff's base formation between periodate-oxidized tRNAPhe (tRNAPheox) and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation. The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA. The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the beta subunit (Mr 63000). The phenylalanine residue, either free or joined to the adenylate, could be cross-linked with equal efficiency to eigher type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits. The Schiff's base formation between tRNAPheox and the enzyme shows the existence of a lysyl group close to the binding site for the 3'-terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast phenylalanyl-tRNA synthetase with pyridoxal phosphate and the 2',3'-dialdehyde derivative of ATP, oATP.

Affinity Labels↗
PubMed1989

A 62 kDa protein is photoaffinity labelled by [3H]felodipine in vascular smooth muscle, but not in cardiac and skeletal muscle.

Irradiation of a cytosolic fraction from vascular smooth muscle in the presence of [3H]felodipine resulted in the labelling of a protein with an apparent molecular weight of 62 kDa. The labelling was seen on UV-irradiation at 360 nm, but not at 254, 278 or at wavelengths above 410 nm. The photolabelling was enhanced in the absence of oxygen. In cytosolic fractions prepared from porcine liver, cardiac and skeletal muscle no photoaffinity labelling of proteins between 90 and 45 kDa could be demonstrated. The results suggest that felodipine is a photoaffinity ligand and that felodipine binds to a soluble protein present in vascular smooth muscle but not in the other tissues tested.

Affinity Labels↗
PubMed1987

Photoaffinity labeling of the beta-adrenergic receptor in synaptic membranes of rat cerebral cortex and cerebellum.

The beta-adrenergic receptor polypeptides in synaptic membranes of rat cerebral cortex and cerebellum have been identified using the photoaffinity label [125I]iodoazidobenzylpindolol. The major receptor polypeptides possess apparent molecular weights of 62,000 and 49,000 (cerebral cortex) or 59,000 and 46,000 (cerebellum). Treatment of the membranes with endoglycosidase F caused the receptor polypeptides from both tissues to exhibit lower apparent molecular weight (51,000) on sodium dodecyl sulfate polyacrylamide gels, indicating that beta-adrenergic receptors in mammalian brain are glycoproteins as are the receptors in peripheral tissues.

Affinity Labels↗
PubMed1990

Photoaffinity labeling of the lysosomal neuraminidase from bovine testis.

ASA-NeuAc2en, a photoreactive arylazide derivative of sialic acid, is shown to be a powerful competitive inhibitor of lysosomal neuraminidase from bovine testis (Ki approximately 21 microM). Photoaffinity labeling and partial purification of preparations containing this lysosomal neuraminidase activity result in specifically and non-specifically labeled polypeptides. Only labeling in a 55 kDa polypeptide is found to be specific, since it could be prevented by the competitive neuraminidase inhibitor NeuAc2en. We conclude that the 55 kDa polypeptide in the bovine testis beta-galactosidase/neuraminidase/protective protein complex contains the catalytic site of neuraminidase.

Affinity Labels↗
PubMedDate not supplied

A fluorescent probe and a photoaffinity labeling reagent to study the binding site of maytansine and rhizoxin on tubulin.

A fluorescent probe (20-demethoxy-20-[3-[[[5-(dimethylamino)naphthalen-1-yl]sulfonyl] amino]propyl]maytansinol 3-isobutyrate, Dan-PDM-3) and a photoaffinity labeling reagent (20-demethoxy-20-[(p-azidobenzoyl)oxy]maytansinol 3-isobutyrate, DABMI) were prepared by derivatization of ansamitocin P-3 (ASMP-3), a maytansinoid. Dan-PDM-3 consists of a tethered dansyl moiety and a maytansinoid moiety. DABMI contains a p-azidobenzoyl group instead of the tethered dansyl moiety of Dan-PDM-3. These compounds were synthesized by reacting 20-demethoxy-20-hydroxymaytansinol-3 isobutyrate (PDM-3) with the corresponding alkyl halide or benzoic acid. Both inhibit tubulin polymerization as potently as ASMP-3 and compete with ASMP-3 for binding to tubulin. The inhibition constants (Ki) of DABMI for the binding to tubulin of rhizoxin and ASMP-3 were 0.54 and 0.36 microM, respectively, which were nearly equal to the dissociation constant (Kd = 0.43 microM) of DABMI measured by the use of [14C]DABMI. The results suggest that Dan-PDM-3 and DABMI interacted with tubulin at the same site as rhizoxin and maytansine. DABMI is irreversibly bound to tubulin upon irradiation. Dan-PDM-3 and DABMI should be useful probes for studying the binding site.

Affinity Labels↗
PubMed1985

Photoaffinity labeling of the GABAA receptor with [3H]muscimol.

Muscimol is one of the most potent agonist ligands at the gamma-aminobutyric acidA (GABAA) receptor. Analysis of its chemical structure showed it to be a candidate for photoaffinity labeling. In practice, UV irradiation at 254 nm both changed the UV spectrum of muscimol and induced an irreversible binding of [3H]-muscimol to rat cerebellar synaptosomal membrane. After 10 min of irradiation, using 10 nM [3H]muscimol, the specific portion of this binding was 270 fmol/mg protein. (Nonspecific binding was defined as that arising in the presence of 1 mM GABA.) Specific binding increased asymptotically up to 100 nM [3H]muscimol. Irradiation of the membranes themselves did not significantly alter the KD or Bmax of reversible [3H]muscimol binding. However, irradiation of [3H]muscimol reduced its capacity subsequently to photolabel the membranes by 86 +/- 3%. Dose-dependent inhibition of binding was observed with muscimol, GABA, and bicuculline methiodide; with 10 nM [3H]muscimol maximum inhibition was 70% of total labeling and the order of potencies of these three compounds was characteristic of labeling to the GABAA receptor. Baclofen, l-glutamate, and diazepam exerted no effect at high concentrations. SDS-PAGE of the photolabeled membranes indicated specific incorporation of radioactivity into two molecular-weight species. One failed to enter the separating gel, implying a molecular weight greater than 250,000 daltons (250 kD). The molecular weight of the other was identified by fluorography to be about 52,000 daltons (52 kD).

Affinity Labels↗
PubMedDate not supplied

Photoaffinity labeling of atrial natriuretic factor analog atriopeptin III with [gamma-32P]8N3GTP.

We have determined that atrial natriuretic factor (ANF) analog atriopeptin III (AP III) is a guanosine 5'-triphosphate (GTP) binding protein as shown by photoaffinity labeling with the GTP-analog [gamma-32P]8N3GTP (azido-GTP). The photo-insertion was saturable at 40 microM azido-GTP. Half-maximal protection from photo-insertion was observed using 40-microM concentrations of the native nucleotide GTP. The photolabeling was inhibited approximately 70% by addition of micromolar concentrations of the divalent cations MgCl2, MnCl2 or CaCl2. The related purine-containing nucleotide adenosine 5'-triphosphate (ATP) was able to elicit half-maximal protection of AP III from photo-insertion with 20 microM azido-GTP at a concentration of 120 microM. No significant protection from photoinsertion was observed with other compounds such as GDP, GMP, cGMP, UTP and pyrophosphate. Additionally, a carboxy-terminal F-R-Y deleted analog of ANF, AP I; reduced and carboxymethylated AP III; and resact, a peptide released from sea urchin eggs, were not photolabeled by azido-GTP. These results demonstrate that the AP III analog of ANF contains a functional GTP binding site that would be saturated at physiological concentrations of GTP. At present, the role of this interaction in mediating the biological effects of ANF is unknown, but we speculate that direct GTP interaction with AP III may be involved in the mechanism of action of this peptide.

Affinity Labels↗
PubMed1988

The GTP-binding peptide of beta-tubulin. Localization by direct photoaffinity labeling and comparison with nucleotide-binding proteins.

The binding site of the guanine moiety of GTP on beta-tubulin was located within the peptide consisting of residues 63-77, AILVDLEPGTMDSVR. The result was obtained using direct photoaffinity labeling, peptide sequencing, and limited proteolysis. Peptides were identified by end-labeling with a monoclonal antibody against beta-tubulin whose epitope was located between 3 and 4 kDa from the C terminus. The sequence of the GTP-binding site is consistent with predictions from other GTP-binding proteins such as elongation factor Tu or ras p21.

Affinity Labels↗
PubMed1995

Mono-, di- and tri-antennary D-galactose ligands as competitive inhibitors and photoaffinity labels of the hexose transporting system in erythrocytes. A model for the irreversible blocking of receptors in cell membranes.

Starting from pentaerythritol, photolabile mono-, di-, and tri-dentate galactose derivatives as well as their 3H-labelled isotopomers were synthesised. The hydrophilic chains linking the 6 position of D-galactose to pentaerythritol consist of 13 atoms in line. The mono-, di- and tri-dentate compounds, although themselves not transported, inhibit in increasing order 14C-D-galactose transport into erythrocytes. On irradiating whole cells in the presence of ligand with 350-nm UV light, these compounds also in increasing order, could irreversibly block the hexose transport system. Irradiation without ligand has no effect. By using the 3H-labelled tridentate galactose compound the hexose transporter (zone 4.5) is specifically radiolabelled, as could be shown in an SDS-PAGE of membrane proteins from erythrocytes previously photoaffinity labelled. Radiolabelling is significantly suppressed in the presence of D-glucose.

Affinity Labels↗
PubMed1993

Determination of the human liver UDP-glucuronosyltransferase 2B4 domains involved in the binding of UDP-glucuronic acid using photoaffinity labeling of fusion proteins.

The interactions between UDP-glucuronic acid and two human liver UDP-glucuronosyltransferase 2B4 peptides (14-150 and 299-446) purified from E. coli as Staphylococcus aureus protein A fusion proteins have been investigated. Photoaffinity labeling with azidonucleotides ([beta-32P]5N3UDP-Glucuronic acid and [beta-32P]5N3UDP-Glucose) and competition experiments with UDP-glucuronic acid and structurally related compounds emphasized the presence of a specific UDP binding site between amino acids 299 and 446. Moreover, competition experiments strongly suggested an interaction between the amino terminal part of the protein and glucuronic acid. It would involve an electrostatic bond in the binding of the cosubstrate via the carboxyl group of UDP-glucuronic acid and a positively charged amino acid of the N-terminal domain of the enzyme.

Affinity Labels↗
PubMed1997

Application of photoaffinity labeling with [11,12-3H]all-trans-retinoic acid to characterization of rat liver microsomal UDP-glucuronosyltransferase(s) with activity toward retinoic acid.

[3H]All-trans-retinoic acid has been shown to be an effective photoaffinity label for microsomal UDP-glucuronosyltransferases. Labeling of rat liver microsomal proteins with [3H]all-trans-retinoic acid and [32P]5-azido-UDP-glucuronic acid has shown that at least one protein in the 50-56 kDa mass range encompassing the UDP-glucuronosyltransferases photoincorporated both probes. The fraction of solubilized microsomal protein eluted from a UDP-hexanolamine affinity column with 50 microM UDP-glucuronic acid contained two protein bands, both of which photoincorporated [3H] all-trans-retinoic acid and were detected on Western blot by anti-UDP-glucuronosyltransferase antibodies. Enzymatic glucuronidation activity toward atRA in the same fraction was enriched five-fold over that of native or solubilized microsomes.

Affinity Labels↗
PubMed1994

Photoaffinity labeling of creatine kinase with 2-azido- and 8-azidoadenosine triphosphate: identification of two peptides from the ATP-binding domain.

Two different analogs of ATP, [gamma-32P]2N3ATP and and [gamma-32P]8N3ATP, were used to photoaffinity label the MM and BB isoforms of rabbit cytosolic creatine kinase. Evidence that photoinsertion was within the ATP-binding domain was as follows: (1) Assays for creatine phosphate production demonstrated that [gamma-32]2N3ATP and [gamma-32P]8N3ATP are substrates for creatine kinase. (2) Enzymatic activity was inhibited by photolabeling with either analog. (3) Saturation of photoinsertion was observed for both analogs. Half-maximal saturation was observed at 5 microM [gamma-32P]2N3ATP or 12 microM (gamma-32P]8N3ATP. (4) Photoinsertion of both probes could be decreased by micromolar levels of ATP. Immobilized Al3+ affinity chromatography and HPLC were used to isolate the peptides modified by these probes. Overlapping sequence analysis of the isolated peptides from the tryptic and chymotryptic digests of the photolabeled MM isoform revealed that [gamma-32P]8N3ATP photoinserted into the peptide region corresponding to Val279-Arg291, whereas [gamma-32P]2N3-ATP photoinserted into Val236-Lys241. The corresponding peptide (Ile279-Arg291 and Val236-Lys241) from the BB isoform were shown to be selectively modified. We conclude that amino acid residues within the peptide regions 236-241 and 279-291 of rabbit cytosolic creatine kinase are localized within the binding domain for the adenine moiety of ATP. The results also demonstrate the effectiveness and selectivity of Al3+ as the chelating agent in immobilized metal affinity chromatography for the isolation of photolabeled peptides as well as its potential to enhance retention of radiolabel during HPLC.

Adenosine Triphosphate↗