Photoaffinity labelling of a nitrobenzylthioinosine-binding polypeptide from cultured Novikoff hepatoma cells
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Site-specific binding of nitrobenzylthioinosine (NBMPR) to plasma membranes of some animal cells results in the inhibition of the facilitated diffusion of nucleosides. The present study showed that nucleoside transport in Novikoff UA rat hepatoma cells is insensitive to site-saturating concentrations of NBMPR. Equilibrium binding experiments demonstrated the presence of high-affinity sites for NBMPR in a membrane-enriched fraction from these cells. In the presence of uridine or dipyridamole, specific binding of NBMPR at these sites was inhibited. When Novikoff UA membranes were covalently labelled with [3H]NBMPR by using photoaffinity techniques, specifically bound radioactivity was incorporated exclusively into a polypeptide(s) with an apparent Mr of 72,000-80,000, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Covalent labelling of this polypeptide was abolished in the presence of excess nitrobenzylthioguanosine (NBTGR) and reduced in the presence of adenosine, uridine or dipyridamole. The apparent Mr of the NBMPR-binding polypeptide in Novikoff UA cells is significantly higher than that reported for corresponding polypeptides in other cell types (Mr 45,000-66,000). When membrane-enriched preparations from S49 mouse lymphoma cells were photolabelled and mixed with labelled NovikoffUA membrane-enriched preparations, gel electrophoresis resolved the NBMPR-binding polypeptides from the two preparations.Keywords:
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AFFINITY labeling for studying the amino acid topography of specific binding sites in biologically important proteins has emerged as an important biochemical technique during the past three decades. Hormone-specific proteins have been the subjects of affinity labeling during the past two decades, beginning with the analysis of the amino acids which constitute the catalytically active site of the enzymes involved in steroid biosynthesis. More recently, hormone-specific receptor and transport proteins have been characterized, and the amino acid compositions of their binding sites have been described by affinity labeling. The reagents used for affinity labeling are analogs of hormones which produce active site-directed irreversible inhibition in the proteins which they attack. Therefore, they have been regarded as potential new drugs for controlling fertility, treating endocrine disorders, and for treating hormone-sensitive cancers. The three major categories of affinity labeling hormone analogs have been classified according to the reagent groups they possess and the mechanisms by which they inhibit the proteins. Affinity alkylating analogs possess a reagent group which at all times is reactive toward certain amino acids. Photoaffinity labeling ana- logs are only reactive while they absorb light energy, and in the energized state they can react with most amino acids. Autoinactivating substrates have thus far only been used with enzymes because they are reactive toward amino acids only after an enzyme has converted the substrates to a reactive form. All three categories of affinity labeling hormone analogs cause irreversible inhibition when they react with an amino acid at a binding site of a hormone-specific protein.
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ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPhotoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5Anthony De Waal, Luitzen De Jong, Aloysius F. Hartog, and Albertus KempCite this: Biochemistry 1985, 24, 23, 6493–6499Publication Date (Print):November 1, 1985Publication History Published online1 May 2002Published inissue 1 November 1985https://pubs.acs.org/doi/10.1021/bi00344a028https://doi.org/10.1021/bi00344a028research-articleACS PublicationsRequest reuse permissionsArticle Views48Altmetric-Citations17LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
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A systemic approach has been taken in the preparation and evaluation of photoaffinity labeling agents for the estrogen receptor from rat and lamb uterus. Several derivatives of estradiol and the nonsteroidal estrogen, hexestrol, containing photoreactive diazocarbonyl or azide functions have been synthesized. The receptor binding affinity of these compounds and their capacity to photointeract with the estrogen binding site (inactivate) can be assayed inderectly by competition assays. Several of the compounds that showed both resonably high binding affinities and inactivation efficiencies have been prepared in high specific activity, tritium-labeled form. Direct binding measurements with these derivatives in unpurified rat uterine receptor preparations, show that while these compounds bind to the receptor, they also show considerable nonspecific binding to nonreceptor proteins. Irradiation of these derivatives in rat uterine cytosol preparations results in incorporation of large amounts of radioactivity into protein in a covalent fashion. The amount of nonspecific labeling is so large, however, that estrogen site specificity (indicated by protection with unlabeled estradiol) cannot be demonstrated. More recently, we have used a partially purified receptor preparation from lamb uterus. The receptor in this preparation has been disaggregated by mild trypsinization and can be electrophoresed in native form. Electrophoretic analysis of the proteins in photolabeled preparations show some covalent incorporation into the receptor region of the gel with one derivative but not with another. The effectiveness of the photoaffinity labeling reagents prepared thus far is assessed, and suggestions are made for the design of new, more effective reagents.
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Photoaffinity labelling probes, OPALs, were rationally designed and synthesized to identify interacting protein targets of 1,3,4-oxadiazin-5(6H)-one (LL-2003). The labelling strategy enabled successful photo-crosslinking with target proteins in both insect cell lysates and mammalian live cells, demonstrating the efficient and selective labelling of Src. A LC-MS/MS analysis of the IGF-1R overexpressed insect proteome labelled by OPALs identified the binding location of the synthesized probes. More information can be found in the Communication by Jeeyeon Lee et al. on page 1626 in Issue 9, 2019 (DOI: 10.1002/ajoc.201900258).
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The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3 ) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[ 3 H]pyridaben ([ 3 H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron–sulfur cluster N2 to quinone.
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8-Azido-adenosine 5'-triphosphate (n8(3)ATP) appeared to be a suitable photoaffinity label for the protein kinase dependent on adenosine 3':5'-monophosphate (cAMP). It competes with ATP for the high-affinity ATP site in the undissociated form of the kinase and in the phosphotransferase reaction catalyzed by the catalytic subunit. Furthermore, it is accepted as a substrate in the phosphotransfer reaction. n8(3)ATP incorporated into the holoenzyme is covalently bound irradiation. Protection experiments with ATP indicated that this covalent attachment occurs in the high-affinity ATP site of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that n8(3)ATP is bound to the catalytic subunit. After irradiation the enzyme was dissociated by cAMP. Proportional to the incorporated [gamma-32P]n8(3)ATP, a loss in phosphotransferase activity was found. These results support our model that both ATP sites coincide with respect to their adenine binding part. Thus binding of the regulatory subunit to the catalytic subunit would then transform the low-affinity catalytically active ATP site into a high-affinity inactive site.
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