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PubMed1992

Photoaffinity labelling of glycosyltransferases.

The photoaffinity analogues 5-azido-UDP-glucose and 5-azido-UDP-glucuronic acid have proven to be valuable biochemical tools in the studies of nucleoside diphosphate sugar-utilizing enzymes, especially membrane-associated glycosyltransferases. A summary of the past and current uses of these analogues is presented, as well as photoaffinity data for the enzyme UDP-glucose: dolichylphosphate glucosyltransferase (Glc-P-Dol synthase). This enzyme has served as a model membrane-associated glycosyltransferase for demonstrating the uses of 5-azido-UDP-glucose. The advantages of using photoaffinity analogues for the purification and characterization of glycosyltransferases are presented, as well as an outline of the general procedures which can be used in conjunction with these analogues.

Affinity Labels↗
PubMed1985

Interspecies conservation of retinal guanosine 5'-triphosphatase. Characterization by photoaffinity labelling and tryptic-peptide mapping.

Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a process which then triggers the replacement of GDP for GTP by a retinal guanosine 5'-triphosphatase referred to as 'transducin'. The transducin-GTP complex then switches on the phosphodiesterase [Fung, Hurley & Stryer (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 152-156]. The bovine transducin consists of an alpha-subunit (39000 Mr), which is a GTP-binding component, together with a beta-(37000 Mr) and a gamma-subunit (10000 Mr). We have purified retinal transducin from cow, pig, chick and frog. The enzyme specific activities and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic profiles indicate that this enzyme is similar in all species except the frog. Whereas the bovine, pig and chick transducins consist of major 37000- and 39000-Mr components, that of the frog consists of a single 75000-Mr component. Labelling of the GTP-binding components with the photoaffinity label 8-azidoguanosine [gamma-32P]triphosphate demonstrated that the 37000-Mr components of the cow, pig and chick and the 75000-Mr component of the frog were major GTP-binding components. In addition, peptide maps of radioiodinated tryptic peptides indicate that the frog 75000-Mr protein is highly related to the pig transducin. These results demonstrate evolutionary conservation of retinal transducin and the presence of a higher-Mr, but nonetheless highly conserved form, of transducin in the frog. The relationship of this component to the recently reported rod-outer-segment inhibitor protein [Yamazaki, Stein, Chernoff & Bitensky (1983) J. Biol. Chem. 258, 8188-8194] is discussed.

Animals↗
PubMed1991

Identification of 1,4-dihydropyridine binding regions within the alpha 1 subunit of skeletal muscle Ca2+ channels by photoaffinity labeling with diazipine.

To identify regions that are involved in the formation of the dihydropyridine receptor site of skeletal muscle L-type Ca2+ channels, the alpha 1 subunit of the channel complex was specifically labeled with the 1,4-dihydropyridine-receptor-selective photoaffinity probe [3H]diazipine. Photoaffinity-labeled regions were identified by probing labeled proteolytic fragments with several anti-peptide antibodies recognizing different segments of the alpha 1 sequence. Forty to 50% of the alpha 1-associated [3H]diazipine label was contained in the tryptic fragment between Arg-988 and Ala-1023 derived from the loop between segments S5 and S6 in domain III. This region corresponds to a portion of the channel that is believed to contribute to formation of the transmembrane pore. Twenty to 30% of the labeling occurred in a V8 protease fragment between Glu-1349 and Trp-1391. This fragment contains transmembrane segment S6 of domain IV and has previously been shown to form part of the drug receptor for phenylalkylamine Ca2+ antagonists. Our data suggest that the dihydropyridine receptor is formed by close apposition of two discontinuous regions of the alpha 1 subunit sequence in domains III and IV. In light of previous work localizing this receptor site to the extracellular surface of the lipid bilayer, it is proposed that amino acid residues at the extracellular surface in the loop connecting segments IIIS5 and IIIS6 and at the extracellular end of segment IVS6 contribute to formation of the dihydropyridine receptor site.

Affinity Labels↗
PubMed1982

Photoaffinity labeling of the beta-adrenergic receptor with azide derivatives of iodoccyanopindolol.

Two photosensitive iodocyanopindolol derivatives, 1-(4-azidobenzimidyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-1) and 1-(4-azidobenzoyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-2) have been prepared. [125I]ICYP-azide-1 and -2 (specific radioactivity up to 2.2 Ci/mumol) bind specifically and with very high affinity (KD = 40-45 pM) to beta-adrenergic receptors of turkey erythrocyte membranes. When [125I]ICYP-azide-1 or -2 were incubated with membranes and UV-irradiated, two polypeptides (Mr = 40,000 and 50,000) were specifically photolabeled as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides may represent subunits of the beta-adrenergic receptor. The yield of specific covalent label incorporation into both polypeptides was up to 17.2% with [125I]ICYP-azide-2 when expressed as fraction of total beta-receptor binding sites. Since the Mr = 40,000 polypeptide was labeled predominantly and since covalent incorporation had the same concentration dependence as reversible specific binding, this polypeptide could contain a beta-adrenergic ligand binding site. Due to the low working concentration (10-100 pM) of [125I]ICYP-azide-1 and -2, nonspecific labeling of membrane proteins was extremely low. The new photoaffinity labels should therefore become valuable tools for probing beta-receptor structure.

Affinity Labels↗
PubMed1998

Photoaffinity labeling of oxidosqualene cyclase and squalene cyclase by a benzophenone-containing inhibitor.

A new orally active oxidosqualene:lanosterol cyclase (OSLC) inhibitor (Ro48-8071; Morand, O. H. et al. (1997) J. Lipid Res. 38, 373-390) showed potent noncompetitive inhibition of bacterial squalene:hopene cyclase (SHC) from Alicyclobacillus acidocaldarius (IC50 = 9.0 nM, KI = 6.6 nM) and OSLC (IC50 = 40 nM, KI = 22 nM for homogeneous rat liver OSLC). A tritium-labeled isotopomer (18.8 Ci/mmol) of this nonterpenoid inhibitor, which possesses a benzophenone (BP) photophore, was chemically synthesized as a photoaffinity label. Specific, efficient covalent modification of both OSLC and SHC enzymes was observed after UV irradiation at 360 nm. Labeling of both OSLC and SHC by [3H]Ro48-8071 was competitively displaced by coincubation with a 1000-fold molar excess of 18-thia-2, 3-oxidosqualene or the nonterpenoid inhibitor BIBX79. Displacement of labeling of OSLC was also achieved with the suicide substrate (3S)-29-methylidene-2,3-oxidosqualene. Thus, the nonsubstrate Ro48-8071 and both terpenoid and nonterpenoid inhibitors of these enzymes appear to share a common binding site.

Animals↗
PubMed1989

Photoaffinity labelling of gonadotropin releasing hormone binding sites in human epithelial ovarian carcinomata.

A photoaffinity labelled derivative of [D-Lys6]-GnRH was prepared with a bifunctional photolabile reagent (4-azidobenzoyl)-N-hydroxysuccinimide. In rat pituitary membranes, this analog retained high binding affinity (Ka = 0.12 x 10(9) M-1) consistent with a single class of receptors. The analog was iodinated and used for the identification of GnRH binding sites in human epithelial ovarian carcinomata. By sodium dodecyl sulfate electrophoresis in 10% polyacrylamide gel the presence of two labelled components could be demonstrated: a high molecular weight component of 63,200 and a smaller component of 46,000. Competition experiments with unlabelled ligand suggest that it is the high molecular weight component which specifically binds GnRH.

Adenocarcinoma↗
PubMed1980

Mutagenesis by photoaffinity labeling using selected azidofluorenes.

Several 2-azidofluorenes have been synthesized for use as photoaffinity labels inside bacteria. In the dark they were not mutagenic for any Salmonella typhimurium tested. When photolyzed inside the bacteria, all were mutagenic for strain TA1538 to varying degrees, and were considerably less mutagenic in the corresponding repair positive TA1978. None were mutagenic for strain TA1535 or TA1537, although most compounds were toxic for those strains when photolyzed.

Affinity Labels↗
PubMed1982

Photoaffinity labelling of the serotonin carrier protein in platelets and brain synaptosomes.

Azidoimipramine, a photoaffinity labelling reagent for the serotonin transport protein, was synthesized. This reagent, upon irradiation, binds covalently to brain synaptosomes preparation and to gel-filtered platelets. Two-dimensional SDS-polyacrylamide gel electrophoresis-isoelectric focusing and tritium fluorography analysis indicate that two synaptosomal proteins and four platelets proteins were labelled by [3H]azidoimipramine.

Affinity Labels↗
PubMed1984

Photoaffinity labelling of mitochondrial NADH dehydrogenase with arylazidoamorphigenin, an analogue of rotenone.

A photoaffinity-labelling analogue of the respiratory inhibitor rotenone was synthesized from the naturally occurring rotenoid amorphigenin. The analogue inhibits NADH-ubiquinone oxidoreductase activity at concentrations comparable with those of rotenone. Photolysis of the radiolabelled analogue bound to isolated NADH-ubiquinone oxidoreductase resulted in preferential incorporation of radioactivity into a polypeptide of Mr 33 000, particularly at low concentrations of the inhibitor. Preparations of the enzyme differ in a parallel fashion in the content of this polypeptide, the degree of photolabelling by the analogue and their sensitivity to rotenone, providing further evidence that the 33 000-Mr protein forms part of the rotenone-binding site.

Affinity Labels↗
PubMed1986

Separation and proteolytic mapping of the two [3H]cytochalasin B photoaffinity labeled D-glucose-sensitive proteins in the chicken embryo fibroblast plasma membrane.

Photoaffinity labeling with [3H]cytochalasin B detects two D-glucose-sensitive proteins in the chicken embryo fibroblast (CEF) plasma membrane, which accumulate under conditions of glucose starvation and are probably involved in the glucose transport system (Pessin, J.E., et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2286-2290). The two labeled components, designated as peak I (Mr 45,000) and II (Mr 52,000) components, were separated by preparative gel electrophoresis in the presence of sodium dodecyl sulfate. The fractions were digested with S. aureus V8 or papain, and the radioactive products were analyzed by one-dimensional gel electrophoresis. The peptide maps showed that they have different peptide structures. Peptide maps of authentic actin, a possible contaminant of the peak I fractions, were quite different from those of the peak I component. Rous sarcoma virus-transformed CEF have two components similar as to apparent molecular size and peptide maps to those present in glucose-starved cells. The peak I and II components show minimal affinity to agarose-bound Ricinus communis agglutinin which binds the human erythrocyte glucose transporter quite well. The peak II component was more susceptible to proteolysis than the peak I one or the human erythrocyte glucose transporter. However, the peptide maps of the peak II component were similar to those of the human erythrocyte glucose transporter.

Actins↗
PubMed1985

Photoaffinity labeling of pituitary and gonadal receptors for gonadotropin-releasing hormone.

A radioactive photoaffinity label for the GnRH receptor was prepared by derivatization of radiodinated [D-Lys6] des-Gly10-GnRH N-ethylamide with the heterobifunctional photolabile reagent N-hydroxysuccinimidyl-4-azido-benzoate. This high affinity photoreactive analog was employed for radiolabeling and characterization of pituitary GnRH receptors in rat, rabbit, mouse, sheep, and cow adenohypophyses and gonadal GnRH receptors in the rat ovary and testis. In rat, rabbit, and mouse pituitary glands, analysis of the GnRH receptor-ligand complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed two labeled components, both of which were displaced by unlabeled GnRH agonist and antagonist analogs. The larger receptor component was a relatively broad band, with mol wt in rat, rabbit, and mouse of 59,000 +/- 1,900, 62,000 +/- 700, and 60,000 +/- 800, respectively. In the rat pituitary gland, the larger component was composed of 63,000 and 52,000 mol wt components, of which the latter was more heavily labeled and was predominant in purified pituitary gonadotrophs. The mol wts of the smaller components were 40,000 +/- 1,600, 43,000 +/- 1,200, and 41,000 +/- 1,000, respectively. In bovine and ovine pituitary glands, the photolabeled GnRH receptor was a single band with mol wt of 42,000 +/- 1,200 and 39,000 +/- 500, respectively. In the rat ovary and testis, photolabeled GnRH receptors were similar to those in the rat pituitary gland, with two distinct components of 53,000 +/- 1,000 and 42,000 +/- 1,000 mol wt. These findings demonstrate that the pituitary receptors that mediate similar physiological actions of GnRH in different species possess broadly similar structural properties, with minor variations between species. It is also evident that the divergent actions of GnRH in different tissues of the same species, as in the rat pituitary and gonads, are expressed through receptors of similar structure.

Affinity Labels↗
PubMed1989

Photoaffinity labeling of the lumenal K+ site of the gastric (H+ + K+)-ATPase.

A photoaffinity label for the lumenal K+ site of the gastric (H+ + K+)-ATPase has been identified. Seven azido derivatives based upon the reversible K+ site inhibitor SCH 28080 were studied, one of which, m-ATIP (8-(3-azidophenylmethoxy)-1,2,3-trimethylimidazo[1,2-a] pyridinium iodide), was subsequently synthesized in radiolabeled form. In the absence of UV irradiation, m-ATIP inhibited K+ -stimulated ATPase activity in lyophilized gastric vesicles competitively with respect to K+, with a Ki value of 2.4 microM at pH 7.0. Irradiation of lyophilized gastric vesicles at pH 7.0 with [14C]m-ATIP in the presence of 0.2 mM ATP resulted in a time-dependent inactivation of ATPase activity that was associated with an incorporation of radioactivity into a 100-kDa polypeptide representing the catalytic subunit of the (H+ + K+)-ATPase. Both inactivation and incorporation were blocked in the presence of 10 mM KCl but not with 10 mM NaCl, consistent with interaction at the K+ site. The level of incorporation required to produce complete inhibition of ATPase activity was 1.9 +/- 0.2 times the number of catalytic phosphorylation sites in the same preparation. Tryptic digestion of gastric vesicle membranes, labeled with [14C]m-ATIP, failed to release the radioactivity from the membranes suggesting that the site of interaction was close to or within the membrane-spanning sections of this ion pump.

Affinity Labels↗
PubMed2000

Probing subunit interactions in alpha class rat liver glutathione S-transferase with the photoaffinity label glutathionyl S-[4-(succinimidyl)benzophenone].

Glutathionyl S-[4-(succinimidyl)benzophenone] (GS-Succ-BP), an analogue of the product of glutathione and electrophilic substrate, acts as a photoaffinity label of dimeric rat liver glutathione S-transferase (GST), isoenzyme 1-1. A time-dependent loss of enzyme activity is observed upon irradiation of the enzyme with long wavelength UV light in the presence of the reagent. The initial rate of inactivation exhibits nonlinear dependence on the concentration of the reagent, characterized by an apparent dissociation constant of the enzyme-reagent complex (K(R)) of 99 +/- 2 microM and k(max) of 0.082 +/- 0.005 min(-1). Protection against this inactivation is provided by the electrophilic substrate (ethacrynic acid), electrophilic substrate analogue (dinitrophenol), and product analogues (S-hexylglutathione and p-nitrobenzylglutathione) but not by steroids (Delta(5)-androstene-3,17-dione and 17beta-estradiol-3, 17-disulfate). These results suggest that GS-Succ-BP binds and reacts with the enzyme within the xenobiotic substrate binding site, and this reaction site is distinct from the substrate and nonsubstrate steroid binding sites of the enzyme. About 1 mol of reagent is incorporated into 1 mol of enzyme dimer when the enzyme is completely inactivated. Met-208 is the only amino acid target of the reagent, and modification of this residue in one enzyme subunit of the GST 1-1 dimer completely abolishes the enzyme activity of both subunits. In order to evaluate the role of subunit interactions in the Alpha class glutathione S-transferases, inactive GS-Succ-BP-modified GST 1-1 was mixed with unlabeled, active GST 2-2. The enzyme subunits were dissociated in dilute trifluoroacetic acid and then renatured at pH 7.8 and separated by chromatofocusing into GST 1-1, 1-2, and 2-2. The specific activities of the heterodimer toward several substrates indicate that the loss of catalytic activity in the unmodified subunit of the modified GST 1-1 is the indirect result of the interaction between the two enzyme subunits and that this subunit interaction is absent in the heterodimer GST 1-2.

Animals↗
PubMed1995

Photoaffinity labeling of human lysosomal beta-hexosaminidase B. Identification of Glu-355 at the substrate binding site.

The carbene precursor 3-azi-1-[([6-3H]-2-acetamido-2-deoxy-1-beta-D-galactopyranosyl)thi o -butane (also designated [3H]-1-ATB-GalNAc) has been used as a photoaffinity label for human lysosomal beta-hexosaminidase B (Hex B, EC 3.2.1.52) purified to apparent homogeneity from postmortal liver. [3H]-1-ATB-GalNAc behaved as an active site-directed inhibitor, which bound covalently to Hex B upon photolysis at 350 nm and resulted in 15% inactivation of enzyme activity. Up to 75% of the inactivation of Hex B was prevented by including the competitive inhibitor 2-acetamido-2-deoxy-D-glucono-1,5-lactone in the photoaffinity experiment. Incubation of [3H]-1-ATB-GalNAc with the enzyme followed by irradiation and subsequent separation of the three polypeptides composing the beta-subunit led mainly to labeling of the beta a-polypeptide. Subsequent proteolysis of beta a with trypsin and separation of the resulting peptides by high pressure liquid chromatography yielded one prominently labeled peptide fraction. Edman degradation resulted in the sequence E339ISEVFPDQFIHLGGD-EVEFK359. However, no modified amino acid was detected, indicating that the photoaffinity label was presumably bound to the peptide by a labile ester linkage. This was proven when the radiolabel was almost completely released from the peptide by treatment with aqueous ammonium hydroxide. Simultaneously, Glu-355 was converted into Gln-355, which is located within a region of Hex B that shows considerable homology with the alpha-subunit of human hexosaminidase A and other hexosaminidases from various species.

Affinity Labels↗
PubMed1999

Docking of linear peptide antagonists into the human V(1a) vasopressin receptor. Identification of binding domains by photoaffinity labeling.

A novel photoactivatable linear peptide antagonist selective for the V(1a) vasopressin receptor, [(125)I][Lys(3N(3) Phpa)(8)]HO-LVA, was synthesized, characterized, and used to photolabel the human receptor expressed in Chinese hamster ovary cells. Two specific glycosylated protein species at 85-90 and 46 kDa were covalently labeled, a result identical to that obtained with a previous photosensitive ligand, [(125)I]3N(3)Phpa-LVA (Phalipou, S., Cotte, N. , Carnazzi, E., Seyer, R., Mahe, E., Jard, S., Barberis, C., and Mouillac, B. (1997) J. Biol. Chem. 272, 26536-26544). To identify contact sites between the new photoreactive analogue and the V(1a) receptor, the labeled receptors were digested with Lys-C or Asp-N endoproteinases and chemically cleaved with CNBr. Fragmentation with CNBr, Lyc-C, and Asp-N used alone or in combination, led to the identification of a restricted receptor region spanning the first extracellular loop. The results established that sequence Asp(112)-Pro(120) could be considered as the smallest covalently labeled fragment with [(125)I][Lys(3N(3)Phpa)(8)]HO-LVA. Based on the present experimental result and on previous photoaffinity labeling data obtained with [(125)I]3N(3)Phpa-LVA (covalent attachment to transmembrane domain VII), three-dimensional models of the antagonist-bound receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V(1a) receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone vasopressin.

Amino Acid Sequence↗
PubMed1981

Photoaffinity labeling of the insulin receptor in H4 hepatoma cells: lack of cellular receptor processing.

Photoaffinity labeling techniques were used to identify insulin-binding components of the plasma membrane in insulin-responsive, monolayer-cultured hepatoma cells. The activated, photosensitive reagent, an n-hydroxysuccinimide ester of 4-azidobenzoic acid, was coupled with highly purified insulin, and the hormone derivative was subsequently iodinated, bound to cell surface receptors of intact H4 cells, and photoactivated. Ater dissolution of the cells, labeled proteins were analyzed by SDS/polyacrylamide gel electrophoresis under reducing conditions. The main labeled band exhibited an apparent molecular weight of 130,000. Two minor components of apparent mol wt 95,000 and 40,000 were also identified. Specific labeling of all 3 bands was inhibited by simultaneous incubation of the cells with native insulin, but not by the heterologous hormone, glucagon, prior to photoactivation. Binding of azidobenzoyl insulin to H4 cells was time-dependent, as was the correlated labeling of receptor components. Band-labeling by the photosensitive insulin derivative was totally light-dependent; spontaneous covalent linking of insulin and receptor was not observed. The labeled receptor-related proteins were not degraded by the cells under our experimental conditions.

Affinity Labels↗
PubMed1995

Photoaffinity labeling of antibodies for applications in homogeneous fluoroimmunoassays.

A homogeneous noncompetitive immunoassay based on photoaffinity labeling techniques is described. Using this method, a fluorophore (reporter) can be specifically attached to an antibody in the vicinity of its antigen-combining sites. Upon antigen binding, changes in the fluorescence spectrum of the reporter molecule are often observed. Two fluorophores, pyrene and dansyl, were evaluated for this purpose. Also, this technology is ideal for fluorescence energy-transfer immunoassays that require labeling of the antibody with either a donor or acceptor fluorophore. In such cases, a fluorescent dye can be specifically attached near the antigen-combining site, where it can participate in high-efficiency energy transfer with its complementary fluorophore attached to the antigen.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of the nucleotide binding site of actin.

Rabbit skeletal muscle actin was photoaffinity-labeled by the nucleotide analogue 8-azidoadenosine 5'-triphosphate. In both G-actin and F-actin about 25% covalent incorporation was achieved. The labeled actins were digested with cyanogen bromide, and the labeled peptides were isolated and sequenced. In F-actin the label was bound primarily to Lys-336, while in G-actin the label was bound to Lys-336 or to Trp-356. The results indicate that the nucleotide binding site is near the phalloidin binding site of actin [Vanderkerckhove, J., Deboben, A., Nassal, M., & Wieland, T. (1985) EMBO J. 4, 2815-2818]. The binding of the azido group to Trp-356 in G-actin but not in F-actin may indicate that a change in the conformation of actin occurs in this region.

Actins↗