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 829 records · Page 46Linked to original sources
PubMed1985

Photoaffinity labeling of atrial natriuretic factor receptor in bovine and rat adrenal cortical membranes.

Radioiodinated synthetic atrial natriuretic factor (ANF) bound to a single class of high affinity binding sites in the plasma membrane from bovine adrenal cortex with a KD of 7.4 X 10(-10) M. The binding affinities of related peptides showed close parallelism to their potencies in natriuretic and vasorelaxant activities. Incubation of adrenal membranes with radioiodinated 4-azidobenzoyl ANF or a similar derivative of its analogue followed by photolysis resulted in specific radiolabeling of a protein band in SDS gel electrophoresis with an apparent Mr of 124,000 in bovine or Mr of 126,000 in rat, which was abolished by inclusion of unmodified ANF in the incubation. Prevention of the labeling was dependent on the concentration of ANF and was not observed with atriopeptin I or with unrelated peptides, angiotensin II, ACTH or [Arg8] vasopressin. These results indicate specific covalent labeling of ANF-receptor or its subunit by the photoaffinity ligands.

Adrenal Cortex↗
PubMed1983

Oligomeric structure of muscarinic receptors is shown by photoaffinity labeling: subunit assembly may explain high- and low-affinity agonist states.

The potent muscarinic photoaffinity reagent N-methyl-4-piperidyl p-azidobenzilate (azido-4NMPB) was used to covalently label specific muscarinic binding sites in various brain regions and in the heart. In the cortex and hippocampus, a single specifically labeled protein with an apparent molecular mass of 86,000 daltons was detected by gel electrophoresis. In the medulla pons, cerebellum, and cardiac atria, there was a 160,000-dalton band in addition to the 86,000-dalton polypeptide. Under certain conditions, alkali or hydroxylamine treatment dissociated both macromolecules into a single 40,000-dalton polypeptide. These results suggest that the muscarinic receptor exists in oligomeric forms and that a dimer and tetramer of a basic 40,000-dalton peptide may exist as interconvertible species. We propose a model to explain the biological architecture of the muscarinic receptors and suggest a possible correlation between the azido-4NMPB-labeled polypeptides and the two states of the receptor observed in agonist binding experiments.

Affinity Labels↗
PubMed1983

Photoaffinity labeling of specific alpha-thrombin binding sites on Chinese hamster lung cells.

Binding sites for alpha-thrombin on cultured Chinese hamster lung cells were identified using photoactivatable cross-linking conjugates of diisopropylphosphorofluoridate-inactivated alpha-thrombin. A series of photoaffinity reagents was synthesized that permitted systematic variation of the extent of thrombin modification and of steric factors affecting the ability of the photoaffinity reagent to contact thrombin receptors. The reagents were synthesized with a tritium label to accurately determine the number of photoaffinity molecules linked to each thrombin. Also, they were synthesized with different length spacer arms between the photoreactive cross-linking group (a nitroarylazide) and the end which linked to alpha-thrombin (a succinimide ester). By calculating the percentage of the thrombin surface that would be accessed by modifying it with a fixed molar excess of each reagent, it was possible to select the photoaffinity reagents that would be most effective for cross-linking 125I-labeled diisopropylphosphorofluoridate-inactivated alpha-thrombin to its cellular binding sites. The validity of this selection procedure was confirmed in experiments in which an Mr = 150,000 cellular component was labeled. This component had the properties of a specific binding site for thrombin since labeling was readily competed for by nonlabeled alpha-thrombin. The cross-linking achieved was due to the photoactivatable reagent since no detectable cross-linked complex was formed in the absence of photoactivation or with 125I-labeled diisopropylphosphorofluoridate-inactivated alpha-thrombin that was not conjugated with the photoaffinity reagent.

Affinity Labels↗
PubMedDate not supplied

Synthesis and characterization of novel spin-labeled photoaffinity nonnucleoside analogues of ATP as structural and EPR probes for myosin.

Two new spin-labeled photoreactive nonnucleoside ATP analogues, 1-(4-azido-2-nitrophenyl)amino-3-(1-oxyl-2,2,5, 5-tetramethylpyrrolidinyl-3-carbamido)-2-propyl triphosphate (SL-NANTP) and 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6, 6-tetramethyl-4-piperidylidene)di(oxymethylene) ethyl triphosphate (SSL-NANTP), were synthesized and characterized. This study aims to develop a second generation of NANTP-based analogues containing immobile spin labels that can be used to monitor conformational changes in myosin during the contractile cycle of muscle. Previous studies have shown that both a photoaffinity nonnucleoside ATP analogue, 2-[(4-azido-2-nitrophenyl)amino] ethyl triphosphate (NANTP) [Nakamaye et al. (1985) Biochemistry 24, 5226-5235], and a photoaffinity ATP analogue, 3'(2')-O-4-[4-oxo-(4-amino-2,2,6, 6-tetramethyl-piperidino-1-oxyl)-4-benzoyl] benzoyl adenosine 5'-triphosphate (SL-Bz(2)ATP) [Wang et al. (1999) J. Muscle Res. Cell Motil. 20, 743-753], behave like ATP in their interactions with myosin. Remarkably, photolabeled myosin recovers all of its normal enzymatic properties after treatment with actin in the presence of MgATP [Luo et al. (1995) Biochemistry 34, 1978-1987]. For SL-NANTP, the spin label moiety is attached to NANTP via an aminomethyl side chain. In SSL-NANTP, attachment is via a restricted spiro ring. The two new probes interact with myosin subfragment-1 (S1) in a manner analogous to ATP, and after photoincorporation, labeled S1 recovers full activity after treatment with actin and MgATP. The electron paramagnetic resonance (EPR) spectrum resulting from S1 photolabeled with SL-NANTP shows a very high degree of probe mobility. However, the EPR spectrum of S1 photolabeled with SSL-NANTP shows that the probe is highly immobilized with respect to S1, constrained to move within a cone of angle 52 degrees (full-width, half-max). Unlike the parent, NANTP, which photolabels on the 23 kDa tryptic fragment of S1, SSL-NANTP photolabels on the 20 kDa fragment. Its highly immobile nature means that it is potentially a useful reporter group to monitor cross-bridge motion in muscle fibers.

Actins↗
PubMed1986

Photoaffinity labelling of dopamine D2 receptors by [3H]azidomethylspiperone.

We have characterized the dopamine D2 receptor photoaffinity probe, [3H]azido-N-methylspiperone ([3H]AMS). In the absence of light, [3H]AMS bound reversibly and with high affinity (Kd 70 pM) to sites in canine striatal membranes and was competitively inhibited by dopaminergic agonists and antagonists with an appropriate D2 receptor specificity. Upon photolysis, [3H]AMS covalently incorporated into a peptide of Mr 92,000 as assessed by fluorography following SDS-polyacrylamide gel electrophoresis. Labelling of this peptide was specifically and stereoselectively blocked by D2 antagonists and agonists. Minor specifically labelled peptides of Mr 70,000-55,000 were observed under some conditions and were the result of proteolytic degradation of the peptide at Mr 92,000.

Affinity Labels↗
PubMed1990

Photoaffinity labelling of the purine-cytosine permease of Saccharomyces cerevisiae.

8-Azidoadenine was used as a photoaffinity reagent to characterize the purine-cytosine permease of Saccharomyces cerevisiae. It is a potent competitive inhibitor of cytosine uptake and irradiation of the cells incubated with the label induced the irreversible inactivation of cytosine uptake. Addition of excess cytosine prevented this labelling which was restricted to the outer face of the plasma membrane since it was not accumulated by the cells. In the strain with the amplified purine-cytosine permease gene the maximum cytosine uptake rate was increased 4-5-fold relative to wild type without a modification of the Michaelis constant of uptake (Kt); no uptake could be measured in the deleted strain. The relative amounts of specific labelling determined for the cells and for membrane preparations were 0, 1 and 4 for the null, the wild-type and the amplified strains, respectively. One major band specifically labelled by [3H]azidoadenine, corresponding to a polypeptide with an apparent molecular mass of 45 kDa, was observed in the wild type, amplified in the strain carrying the multicopy plasmid and not detected in the deleted strain. Therefore this polypeptide corresponds to the purine-cytosine permease.

Adenine↗
PubMed2002

Identification of ligand-binding regions of P-glycoprotein by activated-pharmacophore photoaffinity labeling and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

Energy dependent efflux pumps confer resistance to anticancer, antimicrobial, and antiparasitic drugs. P-glycoprotein (Pgp, ABCB1) mediates resistance to a broad spectrum of antitumor drugs. Compounds that themselves are nontoxic to cells have been shown to act as inhibitors of Pgp. The mechanism of binding and transport of low-molecular-mass ligands by Pgp is still incompletely understood. This study introduces a series of propafenone-related photoaffinity ligands, which combine high specificity and selectivity for Pgp with high labeling efficiency. Molecules are intrinsically photoactivatable in the arylcarbonyl group, which represents a pharmacophoric substructure for this group of ligand molecules. A detailed study of the structure-activity relationship for this type of photoligand is presented. In subsequent experiments, these ligands were used to characterize the drug-binding domain of propafenone-type analogs. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry shows that propafenone-type ligands preferentially label fragments assigned to putative transmembrane segments 3, 5, 6, 8, 10, 11, and 12. Labeled fragments are also identified in a highly charged region of 15 amino acids in the second cytoplasmic loop. This region corresponds to the so-called EAA-like motif, which has been proposed to play a role in the interaction between transmembrane domain and nucleotide binding domain of peroxisomal ATP-binding cassette transporters. In addition, a region in cytoplasmic loop 3 and between TM12 and the N terminus of the Walker A sequence of NBD2 are labeled by the ligands. Therefore, a number of confined protein regions contribute to the drug-binding domain of propafenone-type analogs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

The tonoplast-associated citrate binding protein (CBP) of Hevea brasiliensis. Photoaffinity labeling, purification, and cloning of the corresponding gene.

A detailed comparison of citrate uptake into the vacuole-like lutoids of rubber tree (Hevea brasiliensis Muell. Arg.) and of malate and citrate transport into barley (Hordeum vulgare L.) vacuoles revealed very similar transport specificities. In order to identify proteins mediating the transport, two photoreactive analogues (N'-(2-hydroxy-5-azido)-diazo-N-3,5-benzenedicarboxylic acid and 5-azidoisophthalic acid) of malate/citrate were synthesized and found to efficiently inhibit citrate uptake into barley vacuoles (Ki = 18 microM) and Hevea lutoid vesicles (Ki = 27 microM). In vacuoles from both plant species, these photoaffinity probes specifically labeled a single protein with a molecular mass of 23.6 kDa. This citrate binding protein (CBP) was purified to homogeneity from Hevea lutoids, and amino acid sequences were determined for NH2-terminal and tryptic peptides. Using degenerate oligonucleotides of the NH2-terminal sequence, a cDNA coding for the CBP protein of Hevea was isolated. The cDNA codes for a precursor protein of 238 amino acids, containing an NH2-terminal 31-amino acid signal sequence for endoplasmic reticulum targeting, a prerequisite for vacuolar localization. The mature CBP does not show significant sequence similarities to any known primary protein structure and thus represents a member of a novel class of proteins.

Affinity Labels↗
PubMed1989

The receptor for alpha-melanotropin of mouse and human melanoma cells. Application of a potent alpha-melanotropin photoaffinity label.

The melanotropin (MSH) receptor of mouse B16-F1 melanoma cells was characterized by photoaffinity cross-linking, using a potent alpha-MSH photolabel, [norleucine4, D-phenylalanine7, 1'-(2-nitro-4-azidophenylsulfenyl)-tryptophan9]-alpha-melanotropin (Naps-MSH). Its monoiodinated form, 125I-Naps-MSH, displayed a approximately 6.5-fold higher biological activity than alpha-MSH. Scatchard analysis of the saturation curves with 125I-Naps-MSH revealed approximately 20,000 receptors/B16-F1 cell and an apparent KD of approximately 0.3 nM. Analysis of the cross-linked MSH receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a photolabeled band of approximately 45 kDa occurs in B16-F1, B16-F10, and Cloudman S91 mouse melanoma, as well as in human D10 and 205 melanoma but not in non-melanoma cells. The labeled 45-kDa protein had an isoelectric point of 4.5-4.9 as determined by two-dimensional gel electrophoresis. Treatment of the labeled 45-kDa protein of B16-F1 cell membranes by neuraminidase shifted the band to approximately 42 kDa. A similar band of about 42 kDa was also observed after receptor labeling of B16-W4 cells, a cell line with a decreased number of terminal N-linked neuraminyl residues. These results indicate that the labeled 45-kDa glycoprotein contains terminal sialic acid residues, explaining the low pI of this protein, and that it is characteristic for melanoma cells and hence part of the MSH receptor.

Affinity Labels↗
PubMed1979

The use of arylazido-beta-alanyl-ATP as a photoaffinity label for the isolated and membrane-bound mitochondrial ATPase complex.

The effects of a photoaffinity derivate of ATP, arylazido-beta-alanyl-ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl) adenosine 5'-triphosphate, on submitochondrial particles and the partially purified ATPase complex of beef heart mitochondria have been investigated. In the absence of light the ATP analogue has been found to be a substrate for the E132PA1P1-ATP exchange reaction of submitochondrial particles. When photoirradiated in the presence of arylazido-beta-alanyl-ATP, the ATPase activity and the the the [32P]Pi-ATP exchange reaction are inhibited maximally 80%. Arylazido-beta-alanyl-ATP following photolysis is a noncompetitive inhibitor with respect to ATP while arylazido-beta-alanine, the azido-containing adjunct of the ATP analogue, has no inhibitory effect under the same conditions. The inactivating effect of arylazido-beta-alanyl-ATP is prevented in part by the presence of ATP, or ADP and pyrophosphate. Photolabeling produces a covalent binding of the derivative with the F1ATPase being the major protein labeled. The binding of 0.22 mumol of arylazido-beta-alanyl-ATP/mg of mitochondrial protein is associated with a maximal inhibitory effect. The ATPase activity of the partially purified ATPase complex is also sensitive to photoirradiation in the presence of arylazido-beta-alanyl-ATP. When the ATPase complex is associated with liposomes there is an increase in the specific ATPase activity with a 10-fold increase in Vmax and a 4-fold decrease in KmATP associated with a parallel increase in the apparent affinity and maximal inhibitory effect of the arylazido-beta-alanyl-ATP. The photoinhibition of the ATPase complex in the presence of arylazido-beta-alanyl-ATP results in covalent binding of 1.6 mumol of arylazido-beta-alanyl-ATP/mg of protein. The alpha and beta subunits are the only components of the ATPase complex labeled by the [3H]arylazido-beta-alanyl-ATP. The relationship between the arylazido-beta-alanyl-ATP-labeled sites and the nucleotide binding sites on the mitochondrial ATPase is discussed.

Adenosine Triphosphatases↗
PubMed1998

Photoaffinity labeling of D1 and D5 dopamine receptors.

Although human D1 and D5 dopamine receptors are encoded by distinct genes and share only 50% sequence homology at the amino acid level, their pharmacological properties are identical. Using a selective D1 receptor photoaffinity radioligand, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetrahyd ro-1H-3-benzazepine ([125I]MAB), we have further probed the molecular properties of these receptors in transfected GH4C1 rat pituitary cells. Under reversible, non-covalent binding conditions, [125I]MAB bound to both the D1 and the D5 receptors with identical affinities, dopaminergic selectivity and stereospecificity. Upon photoactivation of the bound [125I]MAB, the label was incorporated into a approximately 64,000 mol. wt protein corresponding to the D1 dopamine receptor. However, there was no specific photoincorporation of the ligand observed in D5 receptors. The lack of [125I]MAB photolabeling of D5 receptors was independent of the cell line chosen, since similar results were obtained using other transfected cells. The data suggest that although both D1 and D5 receptors share structurally similar binding sites, the protein domains around the sites are different. Thus, although there are currently no specific compounds which bind preferentially to D1 or D5 receptors, these receptors can be distinguished from one another by the inability of [125I]MAB to photolabel D5, but not D1, receptors. Such selective targeting of a specific receptor may be useful in understanding the functional importance and/or interaction between closely related members of the same receptor family when co-expressed in the same cell.

Animals↗
PubMed1982

Photoaffinity labeling of Klebsiella aerogenes citrate lyase by p-azidobenzoyl coenzyme A.

p-Azidobenzoyl coenzyme A functions as a linear competitive inhibitor for (3S)-citryl-CoA in the citryl-CoA oxaloacetate-lyase reaction catalyzed by the Klebsiella aerogenes deacetylcitrate lyase complex (Ki = 80 microM; (3S)-citryl-CoA Km = 67 microM). Inactivation is irreversible on photolysis of p-azidobenzoyl-CoA in the presence of the deacetylcitrate lyase complex. Mg2+ is not required for the inactivation. Inactivation is blocked by (3S)-citryl-CoA in the presence of ethylenediaminetetraacetic acid. p-Azidobenzoyl-CoA has no effect on the acetyl-CoA:citrate CoA transferase activity of both the deacetylcitrate lyase complex and its isolated transferase subunit. The stoichiometry of the CoA ester binding has been investigated by the use of p-azido[14C]benzoyl-CoA as a photoaffinity reagent. The labeling is exclusively on the lyase beta subunit of the citrate lyase complex.

Acyl Coenzyme A↗
PubMed1984

Photoaffinity labeling of DNA-dependent RNA polymerase from Escherichia coli with 8-azidoadenosine 5'-triphosphate.

A photoaffinity analogue of adenosine 5'-triphosphate (ATP), 8-azidoadenosine 5'-triphosphate (8-N3ATP), has been used to elucidate the role of the various subunits involved in forming the active site of Escherichia coli DNA-dependent RNA polymerase. 8-N3ATP was found to be a competitive inhibitor of the enzyme with respect to the incorporation of ATP with Ki = 42 microM, while uridine 5'-triphosphate (UTP) incorporation was not affected. UV irradiation of the reaction mixture containing RNA polymerase and [gamma-32P]-8-N3ATP induced covalent incorporation of radioactive label into the enzyme. Analysis by gel filtration and nitrocellulose filter binding indicated specific binding. Subunit analysis by sodium dodecyl sulfate and sodium tetradecyl sulfate gel electrophoresis and autoradiography of the labeled enzyme showed that the major incorporation of radioactive label was in beta' and sigma, with minor incorporation in beta and alpha. The same pattern was observed in both the presence and absence of poly[d(A-T)] and poly[d(A-T)] plus ApU. Incorporation of radioactive label in all bands was significantly reduced by 100-150 microM ATP, while 100-200 microM UTP did not show a noticeable effect. Our results indicate major involvement of the beta' and sigma subunits in the active site of RNA polymerase. The observation of a small extent of labeling of the beta and alpha subunits, which was prevented by saturating levels of ATP, suggests that these subunits are in close proximity to the catalytic site.

Adenosine Triphosphate↗
PubMedDate not supplied

N-(3-azidophenyl)-N-methyl-N'-([4-1H]- and [4-3H]-1-naphthyl)guanidine. A potent and selective ligand designed as a photoaffinity label for the phencyclidine site of the N-methyl-D-aspartate receptor.

A novel radiolabeled photoaffinity ligand has been synthesized for the phencyclidine (PCP) site of the N-methyl-D-aspartate (NMDA) receptor. N-(3-Azidophenyl)-N-methyl-N'-([4-3H]-1-naphthyl)guanidine (13) was prepared with a specific activity of 25 Ci/mmol by diazotization of N-(3-aminophenyl)-N-methyl-N'-([4-3H]-1-naphthyl)guanidine (12) followed by treatment with sodium azide. Guanidine 12 was obtained by catalytic tritiation of N-(4-bromo-1-naphthyl)-N'-methyl-N'-(3-nitrophenyl)guanidine (11). The nontritiated analog 5 of 13 was prepared beginning with N-methyl-N'-1-naphthyl-N-(3-nitrophenyl)guanidine (9). The guanidines 9 and 11 were prepared in moderate yield by the aluminum chloride-catalyzed reaction of N-methyl-3-nitroaniline hydrochloride with 1-naphthylcyanamide and 4-bromo-1-naphthylcyanamide, respectively. Azide 5 showed high selectivity and affinity (IC50 = 100 nM vs [3H]MK801; 3000 nM vs [3H]ditolylguanidine) for the PCP site of the NMDA receptor in guinea pig brain homogenate. Photolabeling experiments with 13, however, failed to radiolabel a significant amount of receptor polypeptide.

Affinity Labels↗
PubMed1985

Photoaffinity labeling of a viral induced protein from tobacco. Characterization of nucleotide-binding properties.

We have used the photoaffinity analogs 8-azidoadenosine 5'-triphosphate (8-N3ATP) and 8-azidoguanosine 5'-triphosphate (8-N3GTP) to investigate the relationship between a viral induced protein (Mr = 120,000) in tobacco mosaic virus (TMV)-infected tobacco and the TMV-induced RNA-dependent RNA polymerase activity. When the radioactive analogs [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP were incubated with the tobacco tissue homogenate from TMV-infected plants, incorporation of label occurred into the viral induced protein in the presence of UV light. The incorporation was found to be totally dependent on UV-illumination and greatly enhanced by Mg2+. Saturation of photoincorporated label indicates an apparent Kd of 16 microM (+/- 3 microM) and 12 microM (+/- 3 microM) for 8-N3ATP and 8-N3GTP, respectively. Protection against photolabeling by [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP with various nonradioactive nucleotides and nucleosides suggests that the photolabeled site is protected best by nucleoside triphosphates. At 200 microM both deoxyribonucleoside triphosphates and ribonucleoside triphosphates were very effective at protecting the site from photolabeling. These data suggest that the photolabeled protein may be part of an RNA-dependent RNA polymerase. The utility of nucleotide photoaffinity analogs as a method to study viral induced nucleotide-binding proteins is discussed.

Adenosine Triphosphate↗
PubMed1987

Preparation and biological evaluation of a potential photoaffinity label for the prostaglandin H2/thromboxane A2 receptor.

Two aromatic azides (24 and 26) were prepared as potential photoaffinity probes for the PGH2/TXA2 receptor. The compounds are based on the well-characterized PGH2/TXA2 receptor antagonist 13-azaprostanoic acid, with the terminus of its lower side chain replaced with phenoxy (24) or benzyl (26) azide functionality. The two compounds were shown to irreversibly inhibit platelet function after photolysis and resuspension. However, of the two aromatic azides, only the benzyl derivative 26 appeared to be selective for the prostaglandin pathway. The latter compound was also prepared as the aromatic 125I (29) derivative, which may ultimately prove useful as a labeled probe for the identification and isolation of the putative TXA2/PGH2 receptor.

Adenosine Diphosphate↗
PubMed1985

Covalent modification of the recA protein from Escherichia coli with the photoaffinity label 8-azidoadenosine 5'-triphosphate.

We have covalently modified the recA protein from Escherichia coli with the photoaffinity ATP analog 8-azido-[alpha-32P]ATP (N3-ATP). Covalent attachment of N3-ATP to recA protein is dependent on native protein conformation and is shown to be specific for the site of ATP hydrolysis by the following criteria. (i) Binding of the probe to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine 5'-O-(thiotriphosphate), ADP, and UTP, but not by adenosine; (ii) N3-ATP is efficiently hydrolyzed by recA protein in the presence of single-stranded DNA; (iii) labeling of recA protein occurs at a single site as judged by two-dimensional thin-layer peptide mapping and high-performance liquid chromatography peptide separation. We have purified and identified a tryptic fragment, spanning amino acid residues 257-280, which contains the primary site of attachment of N3-ATP. This peptide is likely to be contained within the ATP hydrolytic site of recA protein.

Adenosine Triphosphate↗
PubMed1999

Photoaffinity labeling of diphtheria toxin fragment A with 8-azidoadenosyl nicotinamide adenine dinucleotide.

Diphtheria toxin fragment A (DT-A) is an important enzyme in the class of mono(ADP-ribosyl)transferases. To identify peptides and amino acid residues which form the NAD(+) binding site of DT-A using a photoaffinity approach, the photoprobes nicotinamide 8-azidoadenine dinucleotide (8-N(3)-NAD) and nicotinamide 2-azidoadenine dinucleotide (2-N(3)-NAD) were synthesized. Binding studies gave an IC(50) of 2.5 microM for 8-N(3)-NAD and 5.0 microM for 2-N(3)-NAD. Irradiation of DT-A and low concentrations of [alpha-(32)P]-8-N(3)-NAD with short-wavelength UV light resulted in rapid covalent incorporation of the photoprobe into the protein. The photoincorporation was shown to be specific for the active site with a stoichiometry of photoincorporation of 75-80%. After proteolytic digestion of photolabeled DT-A, derivatized peptides were isolated using immobilized boronate affinity chromatography followed by reversed phase HPLC. Radiolabeled peptides originating from two regions of the protein were identified. Chymotryptic digestion produced labeled peptides corresponding to His(21)-Gln(32) and Lys(33)-Phe(53). Lys-C digestion gave overlapping peptides Ser(11)-Lys(33) and Ser(40)-Lys(59). Tyr(27) was identified as the site of photoinsertion within the peptide His(21)-Gln(32) on the basis of the absence of PTH-Tyr at the predicted cycle during sequence analysis and by the lack of predicted chymotryptic cleavage at Tyr(27). Within the second modified peptide Ser(40)-Lys(59), Trp(50) is the most probable site of modification. Identification of Tyr(27) as a site of photoinsertion is in agreement with its placement in the NAD binding site of the X-ray structure of the proenzyme DT-NAD complex [Bell, C. E., and Eisenberg, D. (1996) Biochemistry 35, 1137]. Trp(50) is far from the adenine ring in the crystallographic model; however, site-directed mutagenesis studies suggest that Trp(50) is a major determinant of NAD binding affinity [Wilson, B. A., Blanke, S. R., Reich, K. A., and Collier, R. J. (1994) J. Biol. Chem. 269, 23296-23301].

Amino Acid Sequence↗