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PubMed1985

Regional heterogeneity of rat brain phencyclidine (PCP) receptors revealed by photoaffinity labeling with [3H] azido phencyclidine.

Photoaffinity labeling of rat brain phencyclidine (PCP) receptors with [3H] azido phencyclidine ([3H]AZ-PCP) reveals the existence of five polypeptides which are specifically labeled by the affinity probe (Mr's 90,000, 62,000, 49,000, 40,000 and 33,000). These labeled components are unevenly distributed in rat brain. In the frontal cortex, thalamus and olfactory bulb, the major bands labeled are the Mr's 90 K and 62 K polypeptides; in the cerebellum most of the labeling is in the 90 K and 33 K bands; and in the hippocampus all but the Mr 40 K band are heavily labeled. Together with dexoxadrol/[3H]PCP competition binding data, which indicated the existence of high and low affinity dexoxadrol/PCP binding sites, these results suggest regional heterogeneity of PCP receptors. The regional distribution of the high affinity dexoxadrol binding sites correlates best with that of the Mr 90 K polypeptide.

Affinity Labels↗

PubMed1989

Azido derivatives of dicarboxylic acids for photoaffinity labeling of mitochondrial carriers.

New photoaffinity probes, N-(4-azidosalicylic)-aminosuccinic acid, 3-(4-azidophenylazo)-4-hydroxyphenylmalonic acid, (4-azido-2-nitroanilino)-N-succinic acid, 4-azidophenacylthiosuccinic acid and 4-azidophenylsuccinic acid, were synthesized and characterized chemically. They differ in the distance between dicarboxylic and azido groups, hydrophobicity and acidic moiety. These between dicarboxylic and azido groups, hydrophobicity and acidic moiety. These reagents can be applied for photoaffinity labeling of mitochondrial anion carriers and enzymes interacting with dicarboxylic acids. Inhibition and labeling of the dicarboxylate carrier is presented.

Affinity Labels↗

PubMed1985

Comparative characterization of thyroid hormone receptors and binding proteins in rat liver nucleus, plasma membrane, and cytosol by photoaffinity labeling with L-thyroxine.

Photoaffinity labeling with underivatized thyroxine (T4) was used to identify and compare the T4 binding proteins in rat liver cytosol, nuclear extract, and purified plasma membrane. When these subcellular fractions were incubated with a tracer concentration of [125I]T4, irradiated with light above 300 nm, and individually analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the radioactivity profiles revealed the presence of T4 binding proteins of molecular masses of 70, 52, 43, 37, 30, and 26 kilodaltons (kDa) in cytosol, of 96, 56, 45, and 35 kDa in nuclear extract, and of 70, 44, and 30 kDa in plasma membrane. Competition experiments performed in the presence of a 1000-fold excess of unlabeled T4 demonstrated that these binding proteins display different hormone binding activities. The similar electrophoretic mobilities of some binding proteins present in the different subcellular fractions, i.e., the 70-, 43-45-, and 30-kDa proteins, suggested that these proteins might be identical. However, double-labeling experiments in which plasma membrane, nuclear extract, and cytosol were photolabeled with either [125I] or [131I]T4 and mixed, two at a time, in all possible combinations showed that from one cellular fraction to another, the radioactivity peaks corresponding to the approximately 70-, 43-45-, and 30-kDa proteins were not superimposed. Their relative positions on the gel differed by one or two slices, which indicated differences in molecular mass of 1.9-3.6 kDa. Moreover, enzymatic digestion with Staphylococcus aureus V8 protease of these three proteins, prepared from each subcellular fraction, yielded dissimilar peptide patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed1980

Photoaffinity labeling of corticotropin receptors.

A photoaffinity label for corticotropin (ACTH) receptors was prepared by selective chemical modification of the single tryptophan residue in the hormone by reaction with 2-nitro-5-azidophenylsulfenyl chloride. The photoreactive derivative, [(2-nitro-5-azidophenylsulfenyl)-Trp9]ACTH (2,5-NAPS-ACTH), stimulated corticosterone synthesis to 60% of the maximal rate induced by ACTH in isolated rat adrenocortical cells. 2.5-NAPS-ACTH caused only a marginal stimulation of cyclic AMP production compared to the unmodified hormone. Stimulation of corticosterone production and cyclic AMP accumulation induced by ACTH were both inhibited in a competitive manner by 2,5-NAPS-ACTH. Photolysis of adrenocortical cells in the presence of 2,5-NAPS-ACTH resulted in a 40% inactivation of ACTH receptors mediating steroidogenesis, as shown by the decrease in response to subsequent stimulation with ACTH. No loss of function was observed when photolysis was conducted in the presence of the photoresistant analog [(2,4-dinitrophenylsulfenyl)-Trp9]ACTH. Covalent attachment of the hormone to the receptors was also demonstrated by photolyzing adrenocortical cells in the presence of tritiated 2,5-NAPS-ACTH of high specific radioactivity (90 Ci/mmol) and analyzing the cell proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A protein with an approximate molecular weight of 100,000 was specifically labeled by this procedure. The unique labeling of an adrenocortical cell protein and the concomitant loss of ACTH responsiveness suggest that physiologically relevant receptors are photolabeled by this method.

Adrenal Cortex↗

PubMed1993

Effects of chronic low-level lead exposure on mRNA expression, ADP-ribosylation and photoaffinity labeling with [alpha-32P]guanine triphosphate-gamma-azidoanilide of GTP-binding proteins in neurons isolated from the brain of neonatal and adult rats.

The effects of chronic lead exposure on mRNA expression, ADP-ribosylation and photoaffinity labeling with [alpha-32P]guanine triphosphate-gamma-azidoanilide ([32P]GTP-A) of alpha i or alpha s subunit of G protein were investigated in neurons isolated from the brain of neonatal and adult rats exposed to lead acetate or sodium acetate (for control). Rats were exposed by oral feeding for 10 days or 20 weeks to a low level of lead acetate or sodium acetate. The exposure started either prenatally or at an adult age. The expression of alpha i-mRNA in neurons obtained from the brain of control neonatal rats was significantly higher than that of the expression in samples obtained from the brain of control adult rats or the brain of rats exposed to lead at an adult age. The expression of alpha i-mRNA in neurons obtained from the brain of control neonatal rats, lead-exposed neonatal rats and adult rats prenatally exposed to lead did not differ significantly. Chronic lead exposure did not affect the expression of alpha s-mRNA in neurons obtained from the brain of neonatal and adult rats. The ADP-ribosylation or the photoaffinity labeling with [32P]GTP-A of alpha i or alpha s subunits reflected the developmental pattern of the expression of alpha i or alpha s-mRNA. The incorporation of radioactivity in alpha i-subunit obtained from the brain of control neonatal rats, lead-exposed neonatal rats and rats prenatally exposed to lead was greater than the incorporation in alpha i-subunit obtained from the brain of control adult rats or rats exposed to lead at an adult age. The incorporation of radioactivity did not differ significantly in alpha s-subunits obtained from control or lead-exposed neonatal and adult rats. These observations indicate that (1) the mRNA expression, ADP-ribosylation and photoaffinity labeling with [32P]GTP-A of alpha i-subunit decrease, whereas the mRNA expression, ADP-ribosylation and photoaffinity labeling with [32P]GTP-A of alpha s-subunit do not change as animals age after postnatal day 10, (2) chronic prenatal lead exposure delays the age-dependent decrease in mRNA expression, ADP-ribosylation and photoaffinity labeling of alpha i subunit, and (3) chronic adult exposure does not cause these changes.

Adenosine Diphosphate Ribose↗

PubMed1994

The GLUT3 glucose transporter is the predominant isoform in primary cultured neurons: assessment by biosynthetic and photoaffinity labelling.

Cerebellar granule neurons in primary culture express increasing levels of two glucose transporter isoforms, GLUT1 and GLUT3, as they differentiate in vitro. We have determined the relative abundance of GLUT1 and GLUT3 in these neurons by three different labelling methods. (1) Photoaffinity cell surface labelling of neurons with an impermeant bis-mannose photolabel revealed 6-10-fold more GLUT3 than GLUT1 and dissociation constants (Kd) for the photolabel of 55-68 microM (GLUT3) and 146-169 microM (GLUT1). Binding to both transporters was inhibited by cytochalasin B. (2) Photoaffinity labelling of neuronal membranes with a permeant forskolin derivative showed 5.5-8-fold more GLUT3 than GLUT1, whereas in rat brain membranes containing both neuronal and glial membranes, GLUT3 and GLUT1 were detected in similar proportions. (3) Biosynthetic labelling of neurons with [35S]methionine and [35S]cysteine showed GLUT3 to be 6-10-fold more abundant than GLUT1. Thus GLUT3 is quantitatively the predominant glucose-transport isoform in cultured cerebellar granule neurons.

Affinity Labels↗

PubMed2006

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics.

Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.

Azides↗

PubMed2005

Photoaffinity labeling of P450Cam by an imidazole-tethered benzophenone probe.

[(3)H]4-Benzoyl-N-[2-(imidazole-4-yl)ethyl]benzamide ([(3)H]HBP) was synthesized and used to photoaffinity label P450(Cam). The imidazole moiety of HBP anchors the compound in the P450(Cam) active site by coordination of the heme iron, thereby insuring that covalent modification occurs in the active site. Additionally, the imidazole anchor provides a known binding orientation of HBP to P450(Cam) from which conclusions about enzyme structure can be drawn based upon the locations of photoadducted residues. Two sites of adduction were identified by MS analysis of digested, photoaffinity labeled P450(Cam). Photoaffinity labeling experiments in the presence of the type II competitive inhibitor, 1-phenylimidazole, were used to assess the specificity of the photoadducts characterized. One adduct was located at Met103 on the flexible B'/C loop region of P450(Cam). The other adduct was localized on the C-helix at Met121. The implications of these data are discussed.

Amino Acid Sequence↗

PubMed1996

Photoaffinity labeling of the head-activator receptor from hydra.

A photoaffinity ligand for the head-activator (HA) receptor from hydra was synthesized using solid-phase peptide synthesis and coupling of two HA peptides over their epsilon-amino groups of Lys7 with succinimidyl esters. The new ligand, Bpa-HA-HA bipeptide, contains one normal HA peptide and another where p-benzoylphenylalanine (Bpa) was added at the amino terminus to allow ultraviolet activation and Tyr11 instead of Phe11 for radioiodination. The 125I-Bpa-HA-HA bipeptide bound with nanomolar affinity to the HA receptor from the multiheaded mutant of Chlorohydra viridissima as measured in a filter assay. After photoaffinity labeling of the hydra membrane fraction, a 200-kDa band was detected using reducing or non-reducing SDS/PAGE and autoradiography. Unlabeled HA derivatives, but no other neuropeptides, inhibited the labeling. Competition experiments with HA-HA homobipeptide in the nanomolar range indicate that predominantly the low-affinity and not the high-affinity HA receptor was photolabeled. Further evidence that the labeled molecule is the HA receptor comes from specific photoaffinity labeling with a second ultraviolet-activatable ligand containing p-nitrophenylalanine. The HA receptor could be functionally solubilized with Triton X-100 or Chaps. In the solubilizate the 200-kDa HA receptor was photolabeled specifically by both ligands. Liquid-phase isoelectric focussing of the solubilizate indicated a pI of about 5.4 of the photolabeled molecule. After chemical deglycosylation with trifluoromethanesulfonic acid, the apparent molecular mass of the labeled molecule was decreased to 180 kDa, indicating that the receptor is glycosylated.

Affinity Labels↗

PubMed1993

Use of drug-specific antibodies to identify ethidium adducts produced in Trypanosoma brucei by photoaffinity labeling.

A photoreactive azido analog of the trypanocide ethidium bromide, 3-amino-8-azido-5-ethyl-6-phenylphenanthridinium chloride, attached covalently to calf thymus DNA (CT DNA) by photoaffinity labeling, was used to generate antibodies for the drug analog. The specificity of the antiserum was tested using enzyme-linked immunoadsorbant assays (ELISA) against immobilized antigen (photoaffinity labeled DNA) and by both the avidin-biotin peroxidase reaction and indirect immunofluorescence performed on smears of drug treated trypanosomes. The reaction of the antiserum with the covalently bound drug adduct was diminished effectively by prior incubation with an excess of ethidium monoazide, ethidium diazide, and ethidium bromide, and to a lesser extent by the DNA-ethidium complex, the diazide-DNA or RNA adduct, and the monoazide-RNA adduct. DNA which had been photoaffinity labeled with either the propidium or the acridine moiety did not react. The antiserum recognition of DNA photoaffinity labeled with ethidium monoazide was based on the substituted phenanthridinium ring system of the parent ethidium, as evidenced by competition binding studies involving the free monoazido analog (EA1), the diazido analog (EA2), and the parent compound, ethidium bromide (EB). This approach and the sensitivity it provides should prove useful for identifying the distribution and fate of covalently bound drugs resulting from antiparasitic drug treatment, and for studying their roles in antiparasitic action.

Affinity Labels↗

PubMed1995

Characterization of the arachidonate and ATP binding sites of human 5-lipoxygenase using photoaffinity labeling and enzyme immobilization.

The arachidonic acid and the ATP binding sites of human 5-lipoxygenase were characterized using photoaffinity labeling and immobilization of the enzyme on ATP-agarose. Photoaffinity labeling of the active site of 5-lipoxygenase was achieved with a novel thiopyranoindole inhibitor containing a 4-azido-3-iodobenzenesulfonyl moiety (L-708,714). This probe was found to inhibit the activity of 5-lipoxygenase (IC50 = 0.3 microM) and to covalently label the enzyme after UV light irradiation. The labeling was inhibited by arachidonic acid, N-hydroxyurea, and dihydrobenzofuranol inhibitors which have been shown to reduce the non-heme iron center of 5-lipoxygenase. Photoaffinity labeling of 5-lipoxygenase by L-708,714 was dependent on the presence of both Ca2+ ions and phospholipids and was independent of ATP. It occurred at similar levels using native (Fe2+), oxidized (Fe3+), or H2O2-inactivated enzyme, but was abolished by heat inactivation of the enzyme. Competition of the labeling by various thiopyranoindoles and other inhibitors such as L-697,198,ZD-2138, and zileuton was found to be related to their inhibitory potency. Immobilized 5-lipoxygenase on ATP-agarose was found to be selectively eluted by adenine nucleotides (ATP > ADP > AMP) but not by solutions containing high salt concentrations, mild detergents, arachidonic acid, or inhibitors. 5-Lipoxygenase inhibitors were selectively retained on the immobilized enzyme and eluted by buffer containing arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗

PubMed1984

Direct photoaffinity labeling of the high affinity nitrendipine-binding site in subcellular membrane fractions isolated from canine myocardium.

[3H]Nitrendipine and high intensity ultraviolet irradiation have been used to photoaffinity label the protein component of the high affinity nitrendipine-binding site in subcellular membrane fractions from canine cardiac muscle. Irradiation of isolated cardiac membranes in the presence of [3H]nitrendipine resulted in the covalent labeling of a protein component that migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 32,000. Incorporation of [3H]nitrendipine did not occur in the absence of irradiation. The photoaffinity labeling of the 32,000-Da protein by [3H]nitrendipine was inhibited by excess unlabeled nitrendipine, nifedipine, or verapamil. EDTA, ATP, and La3+, which are known to reduce high affinity nitrendipine binding, also inhibited the photoaffinity labeling of this membrane protein by [3H]nitrendipine. The 32,000-Da [3H]nitrendipine-labeled protein was found to be enriched in the ryanodine-sensitive fraction of cardiac sarcoplasmic reticulum and absent from the ryanodine-insensitive fraction of cardiac sarcoplasmic reticulum which is known to lack high affinity nitrendipine binding. Therefore, the 32,000-Da photoaffinity-labeled [3H]nitrendipine-binding protein exhibits properties identical to those expected for the protein component of the high affinity nitrendipine-binding site in isolated cardiac membranes.

Affinity Labels↗

PubMed2000

Direct photoaffinity labeling of Kir6.2 by [gamma-(32)P]ATP-[gamma]4-azidoanilide.

ATP-sensitive potassium (K(ATP)) channels are under complex regulation by intracellular ATP and ADP. The potentiatory effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We have previously reported that Kir6.2 can be directly labeled by 8-azido-[gamma-(32)P]ATP. However, the binding affinity of 8-azido-ATP to Kir6.2 was low probably due to modification at 8' position of adenine. Here we demonstrate that Kir6.2 can be directly photoaffinity labeled with higher affinity by [gamma-(32)P]ATP-[gamma]4-azidoanilide ([gamma-(32)P]ATP-AA), containing an unmodified adenine ring. Photoaffinity labeling of Kir6.2 by [gamma-(32)P]ATP-AA is not affected by the presence of Mg(2+), consistent with Mg(2+)-independent ATP inhibition of K(ATP) channels. Interestingly, SUR1, which can be strongly and specifically photoaffinity labeled by 8-azido-ATP, was not photoaffinity labeled by ATP-AA. These results identify key differences in the structure of the nucleotide binding sites on SUR1 and Kir6.2.

ATP-Binding Cassette Transporters↗

PubMed1994

Photoaffinity labeling for evaluation of uridinyl analogs as specific inhibitors of rat liver microsomal UDP-glucuronosyltransferases.

The UDP-glucuronosyltransferases (UGT) involved in glucuronidation of endogenous and exogenous toxic compounds transfer the glucuronic acid residue from UDP-glucuronic acid (UDP-GlcUA), to various acceptor groups. A series of compounds that contain N-acyl phenylaminoalcohol derivatives linked to uridine or isopropylideneuridine were tested as UGT inhibitors. The potency of these inhibitors was determined by studying their effect on the photoaffinity labeling of rat liver microsomal UGTs by two photoaffinity probes, [beta-32P]5-azido-UDP-glucuronic acid (5N3UDP-GlcUA) and [beta-32P]5-azido-UDP-glucose (5N3UDP-Glc) and on the enzymatic formation of the two glucuronide conjugates (3-O- and carboxyl-specific) of lithocholic acid. All but one of the compounds tested proved to have an inhibitory effect on UGTs, both in the photoaffinity labeling system and in the enzymatic glucuronidation assay. In the photoaffinity labeling system, the inhibitors containing the isopropylidene moiety were less effective than their unprotected derivatives; however, the protected forms were, with one exception, more potent inhibitors of enzymatic activity. The photoaffinity labeling of UGTs with [beta-32P]5N3UDP-Glc was more susceptible to inhibition by all derivatives than that with [beta-32P]5N3UDP-GlcUA. The effect of one inhibitor, PP50B, on the two enzymatic activities involved in LA glucuronidation was extensively tested. A double-reciprocal plot suggested a competitive inhibition for UDP-GlcUA with an apparent Ki of 35 microM for LA 3-O-glucuronide formation and 94 microM for the carboxyl-linked glucuronide of the same substrate.

Affinity Labels↗

PubMed1978

Fluorescent photoaffinity labeling: adenosine 3',5'-cyclic monophosphate receptor sites.

An approach to the study of protein receptor sites in protein mixtures or supramolecular assemblies by using fluorescence spectroscopy is described. This approach, fluorescent photoaffinity labeling, combines the merits of photoaffinity labeling to attain site-directed reactivity with the probing power of fluorescent ligands. A fluorescent photoaffinity label for cyclic AMP receptor sites of cyclic AMP-dependent protein kinases was synthesized in both unlabeled and radioactive forms. The probe, 8-azido-1,N(6)-ethenoadenosine 3',5'-cyclic monophosphate, mimics cyclic AMP in its ability to stimulate the phosphotransferase activity of the protein kinases and strongly competes with cyclic AMP for its binding sites in all preparations so far tested. Photolysis, after equilibration of protein kinase and 8-azido-1,N(6)-ethenoadenosine 3',5'-cyclic monophosphate in the dark, effects binding of the intermediate nitrene irreversibly and specifically to the cyclic AMP sites with the development of fluorescence. Excess reagent and low molecular weight photolytic products are removable by dialysis. Studies of a crude beef heart preparation containing cyclic AMP-dependent protein kinase suggest that the cyclic AMP binding sites are hydrophobic in nature and strongly immobilize the adenine moiety of the cyclic nucleotide.

Affinity Labels↗

PubMed1994

Evaluation of photoaffinity labeled sites of bacteriorhodopsin using molecular modeling.

Three successful photoaffinity labeling experiments of bacteriorhodopsin (bR) have been reported that used photoactivatible analogs of retinal to label the retinal binding site of the protein. Using molecular modeling techniques, the information about the retinal binding site derived from these studies is compared to the retinal binding site as defined by Henderson et al. (1990) using electron diffraction data. This comparison suggests some limitations to the use of photoaffinity labeling experiments for the determination of high resolution structural information.

Affinity Labels↗

PubMed2000

Deduction of amino acid residues in the GABA(A) receptor alpha subunits photoaffinity labeled with the benzodiazepine flunitrazepam.

Peptide mapping and microsequencing were used to infer the site of photoaffinity labeling by the gamma-aminobutyric acidA receptor modulator [3H]flunitrazepam. Peptide mapping with and without N-deglycosylation was used to restrict the domain for photoaffinity labeling to residues 74-123 of the bovine alpha1 subunit, in agreement with a previously predicted labeling domain between residues 59-148 based on cyanogen bromide fragmentation. Edman degradation of partially purified photolabeled peptides gave release of 3H counts in the ninth cycle of a tryptic peptide sequence. A second V8/chymotryptic peptide produced an impure sequence with release of 3H counts in the seventh through ninth cycle of sequence. The combined data support those previously reported, i.e., that the primary site for photoaffinity labeling by [3H]flunitrazepam is His102 of the bovine alpha1 subunit. In addition we also detected possible secondary labeling of Pro97.

Affinity Labels↗

PubMed1984

Photoaffinity labeling of the stereospecific D-glucose transport system with cytochalasin B.

A method has been developed for photoaffinity labeling components of the hexose transport system with [3H]cytochalasin B. We have demonstrated that UV photoirradiation of intact human erythrocytes or ghost membranes with 0.5 microM [3H]cytochalasin B yielded a broad peak with an Mr of 44,000-70,000. Labeling that was insensitive to the presence of 0.5 M D-sorbitol was substantially inhibited by 0.5 M D-glucose. Approximately 80% of labeling in the region of Mr = 49,000-70,000 was inhibited by the presence of 0.5 M D-glucose, whereas labeling of the 44,000- to 49,000-dalton region was inhibited only 30%. Somewhat different results were obtained from photoaffinity labeling of plasma membranes from chicken embryo fibroblasts (CEF). The [3H]cytochalasin B-labeling patterns had two relatively sharp and discrete peaks at 46,000 and 52,000 daltons. Comparison of plasma membranes from glucose-fed and starved CEF revealed that the total D-glucose-sensitive labeling increased approximately 12-fold in the starved cell membranes. Labeling of the 52,000-dalton polypeptide was more sensitive than that of the 46,000-dalton polypeptide to inhibition by the presence of D-glucose. These results indicate that [3H]cytochalasin B photoaffinity labeling has wide applicability for identifying and covalently binding components of the facilitated hexose transport system.

Affinity Labels↗