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Results for “Photoaffinity Labels”

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PubMed1990

Flunitrazepam photoaffinity labeling of the GABA(A) receptor reduces inhibition of [3H]Ro15-4513 binding by GABA.

The benzodiazepine drugs modulate gamma-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA(A) receptor complex, but which is distinct from the GABA binding site. Ro15-4513 is a benzodiazepine negative modulator of GABA action that displays unique anti-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffinity label nearly 100% of the benzodiazepine binding sites in rat brain homogenates. In contrast, the benzodiazepine positive modulator flunitrazepam photoaffinity labels only 25% of the sites. Here, we have examined the reversible binding of [3H]Ro15-4513, [3H]flumazenil (Ro15-1788), and [3H]flunitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonradioactive flunitrazepam. Photoaffinity labeling with flunitrazepam decreased the subsequent reversible binding of [3H]flunitrazepam and [3H]flumazenil, but increased the binding of [3H]Ro15-4513. The increase in [3H]Ro15-4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent Kd, with no change in Bmax. Following photoaffinity labeling, negative modulation of [3H]Ro15-4513 binding by GABA was lost, whereas positive modulation of residual [3H]flunitrazepam binding was retained. We conclude that the site photoaffinity labeled by flunitrazepam is distinct from the site responsible for reversible binding of [3H]Ro15-4513.

Affinity Labels↗

PubMed1989

Direct photoaffinity labeling of leukotriene binding sites.

Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labeling of leukotriene binding sites. Cryofixation eliminates unspecific labeling taking place in solution by photoisomers and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock-freezing of the samples, irradiation-induced highly reactive excited states and/or intermediates can form covalent bonds with the respective binding site in the frozen state. After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time-dependent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation revealed that only one polypeptide with an Mr of 67,000 was labeled. This polypeptide was identified as albumin. Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent Mr of 48,000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hepatocytes a polypeptide with an apparent Mr of 48,000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent Mr of 25,000, was identified as subunit 1 of glutathione transferases by immunoprecipitation. The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene-metabolizing enzymes, and transport systems.

Animals↗

PubMed1996

Irreversible inhibition of hepatic fatty acid salt uptake by photoaffinity labeling with 11, 11-azistearate.

In order to have a model compound for detection of proteins involved in transport and metabolism of long-chain fatty acid salts by photoaffinity labeling 11,11-azistearate and 11,11-azi[G-3H]stearate (specific radioactivity 2.78 TBq/mmol) were synthesized. The suitability of 11,11-azi[G-3H]stearate for photoaffinity labeling was demonstrated by incorporation into BSA (bovine serum albumin) and H-FABP (hepatic fatty acid salt-binding protein) of rat liver. Repeated photoaffinity labeling resulted in a clear decrease of the binding capacities of both proteins. Labeling of protein mixtures with 11,11-azi[G-3H]stearate showed that binding proteins for long-chain fatty acid salts interact specifically with this probe. Photoaffinity labeling of isolated hepatocytes using 300 microM 11,11-azistearate in the presence of 100 microM BSA resulted in the irreversible inhibition of the uptake of stearate and its analogue 2,2,3,3,18,18,18-heptafluorostearate nearly to the same extent of about 30%. Irreversible inhibition of the uptake of long-chain fatty acid salts by photoaffinity labeling did not alter the mediated transport of cholyltaurine and has no effect on the uptake of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, a compound that crosses the hepatocyte membrane by simple diffusion. The irreversible inhibition of membrane transport by photoaffinity labeling demonstrates the existence of a specific transport system for the uptake of long-chain fatty acid salts into hepatocytes.

Affinity Labels↗

PubMed2003

Construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors.

A new general method, post-photoaffinity-labeling modification (PPALM), for constructing fluorescent saccharide biosensors based on naturally occurring saccharide-binding proteins, lectins, is described in detail. An active-site-directed incorporation of a masked reactive site into a lectin was conducted by using a photoaffinity labeling technique followed by demasking and then chemical modification to yield a fluorescent lectin. Two photoaffinity labeling reagents were designed and synthesized in this study. The labeling reagent with a photoreactive site appended through a disulfide link to a mannoside unit was bound to the saccharide-binding pocket of the lectin concanavalin A (Con A). After light irradiation, the mannoside unit was cleaved by reduction. The unique thiol group thus produced was site-specifically modified with various fluorescent groups (dansyl, coumarin, or dimethylaminobenzoate derivatives) to afford fluorescent Con As. The labeling site was characterized by protease-catalyzed digestion followed by HPLC, MALDI-TOF MS, and tandem mass-mass spectrometry; these methods indicated that the photolabeling step is remarkably site specific. Strong fluorescence was observed in the engineered Con A with a fluorophore, and the emission changed sensitively upon saccharide complexation. The binding constants for various saccharides were determined by fluorescence titration and demonstrated that the binding selectivity and affinity of the engineered Con As are comparable to those of native Con A. The red shift of the emission maximum, the decrease in the fluorescence anisotropy of the dansyl unit, and the increase in the twisted intramolecular charge transfer emission caused by sugar binding to the engineered Con A explicitly indicate that the microenvironment of the appended fluorophores changes from a restricted and relatively hydrophobic environment into a rather freely mobile and hydrophilic environment.

Binding Sites↗

PubMed1995

Photoaffinity labelling of the ATP-binding sites of two Ca2+,Mg-ATPase isoforms in pancreatic endoplasmic reticulum.

Pancreatic rough ER ATP-binding proteins, including two isoforms of SERCA-2b Ca2+,Mg-ATPase, were identified using specific photoaffinity labelling with 8-azido-ATP. 8-Azido-ATP irreversibly inhibited Ca2+,Mg-ATPase activity only after UV irradiation and the inhibition was prevented by inclusion of 5 mM ATP in the labelling reaction. Rough ER proteins of apparent molecular masses 141, 111, 100, 84, 69, 55 and 47 kDa were detected following photoaffinity-labelling with 8-azido-[alpha-32P]ATP. The two bands at 111 kDa and 100 kDa corresponded in molecular mass to the two SERCA-2b Ca2+,Mg-ATPase isoforms previously demonstrated immunologically [1]. Immunoprecipitation of rough ER proteins by a SERCA-2b-specific antibody showed that the two ATPase bands were photoaffinity-labelled. Photoaffinity labelling of the 111 and 100 kDa proteins was: (a) abolished when Ca2+,Mg-ATPase activity was inactivated by EDTA-treatment of rough ER membranes; (b) inhibited by the Ca2+,Mg-ATPase inhibitor vanadate; (c) not affected by thapsigargin. The data demonstrate that pancreatic rough ER contains two isoforms of the SERCA-2b Ca2+,Mg-ATPase whose ATP-binding properties are susceptible to inhibition by vanadate but not thapsigargin.

Adenosine Triphosphate↗

PubMed2001

Photoaffinity labeling of mutant neurokinin-1 receptors reveals additional structural features of the substance P/NK-1 receptor complex.

Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated, photoreactive derivatives of SP containing p-benzoyl-L-phenylalanine (Bpa) in positions 4 and 8, respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa8-SP resulted in a marked reduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinity labeling of the M174A mutant using radioiodinated Bpa4-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2) loop sequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located alpha-helix, extending from Pro175 to Glu183, as well as smaller alpha-helices at the termini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central alpha-helix, approximately 11 A apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.

Amino Acid Sequence↗

PubMed1996

Photoaffinity labeling of human recombinant sulfotransferases with 2-azidoadenosine 3',5'-[5'-32P]bisphosphate.

Photoaffinity labeling with 2-azidoadenosine 3', 5'-[5'-32P]bisphosphate was used to identify and characterize adenosine 3',5'-bisphosphate-binding proteins in human liver cytosol and recombinant sulfotransferase proteins. The sulfotransferases investigated in these studies were the human phenol sulfotransferases, HAST1, -3, and -4, dehydroepiandrosterone sulfotransferase, and estrogen sulfotransferase. The cDNAs for these enzymes have been previously cloned and expressed in COS-7 cells or Escherichia coli. Photoaffinity labeling of all proteins was highly dependent on UV irradiation, was protected by co-incubation with unlabeled adenosine 3',5'-bisphosphate and phosphoadenosine phosphosulfate, and reached saturation at concentrations above 10 microM. To verify that the 31 35-kDa photolabeled proteins were indeed sulfotransferases, specific antibodies known to recognize human sulfotransferases were used for Western blot analyses of photolabeled proteins. It was shown unequivocally that the proteins in the 31-35-kDa region recognized by the antibodies also photoincorporated 2-azidoadenosine 3',5'-[5'-32P]bisphosphate. This is the first application of photoaffinity labeling with 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for the characterization of recombinant human sulfotransferases. Photoaffinity labeling will be also useful in the purification and functional identification of other adenosine 3',5'-bisphosphate-binding proteins and to determine amino acid sequences at or near their active sites.

Adenosine Diphosphate↗

PubMed1984

Photoaffinity-labeled hapten-binding T-cell receptor on a suppressor T-cell hybridoma.

A T-cell hydridoma, 7C3-13-Ag6, which produces a (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific suppressor T-cell factor associated with an I-J determinant, was utilized to study the hapten-binding receptor of T-cells. This hybridoma had been shown to express NP-binding receptor molecules on the cell surface with heteroclitic fine specificity for a cross-reactive hapten, (4-hydroxy-5-iodo-3-nitrophenyl) acetyl (NIP). The stoichiometric analysis of the hapten binding by 7C3-13-Ag6 cells was performed by the measurement of direct binding of highly radioactive haptens to the cell surface. The affinity constant (Ka) of the receptor for N125IP-epsilon-aminocaproic acid (N125IP-cap), as calculated from a Hill plot, was 5.75 X 10(7) M-1 [Hill coefficient (a) = 0.86; expression of receptor sites per cell = approximately 1 X 10(3) on average]. The receptor molecule was specifically affinity labeled with photoreactive nitroaryl azide derivatives of N125IP (510-570 Ci/mmole). The specificity of photoaffinity labeling was demonstrated both by competitive inhibition of labeling with NIP- or NP-cap and by differential photoaffinity labeling based on the reversibility of hapten-receptor interaction. The gel electrophoretic analysis of the photoaffinity-labeled molecule indicated that the hapten-binding receptor of 7C3-13-Ag6 has a mol. wt of 28,000 +/- 3000 and an isoelectric point of 5.6-5.7. No immunoglobulin determinants were detected on the molecule. A comparative immunoprecipitation analysis of the membrane lysate of 7C3-13-Ag6 with monoclonal anti-I-J reagents identified a separate I-J molecule of 25,000 +/- 1000 mol. wt that is distinct from the photoaffinity-labeled hapten-binding molecule.

Affinity Labels↗

PubMed1985

Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5.

The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.

Affinity Labels↗

PubMed1996

A photolabile 2',3'-dideoxyuridylate analog bearing an aryl(trifluoromethyl)diazirine moiety: photoaffinity labeling of HIV-1 reverse transcriptase.

In order to develop a photoaffinity labeling reagent for DNA polymerases, including retroviral reverse transcriptase (RT), we utilized 2',3'-dideoxy-E-5-[4-(3-trifluoromethyl-3H-diazirin-3-yl) styryl]UTP (TDSddUTP) as a substrate dTTP analog. Photoaffinity labeling experiments with human immunodeficiency virus type-1 (HIV-1) RT using a radioactive labeling reagent ([gamma-32P]TDSddUTP) and poly(A).oligo(dT) as the template/primer yielded different results depending on the concentration of Mg2+. In the presence of 0.025 mM Mg2+, photoaffinity labeling showed that TDSddUTP bound selectively to the dTTP binding site in the 66 kDa subunit of the p66/p51 heterodimeric enzyme protein when irradiated by near-UV light (365 nm). In the presence of 4 mM Mg2+ or 0.05 mM Mn2+, TDSddUTP was incorporated into the 3'-end of the primer strand due to RT activity and the resulting photolabile primer bound to the 66 kDa subunit of HIV-1 RT on photoirradiation. These results suggest that TDSddUTP could be a useful tool for studying the substrate binding site(s) of DNA polymerases, including HIV-1 RT, which show affinity for this compound.

Affinity Labels↗

PubMed1981

Isolation and characterization of a photoaffinity-labeled peptide from the catalytic site of prenyltransferase.

Previously we presented evidence for the selective modification of the catalytic site of prenyltransferase by photoaffinity labeling with o-azidophenylethyl pyrophosphate [Brems, D. N., & Rilling, H. C. (1979) Biochemistry 18, 860]. In the present work, we report the isolation and characterization of a CNBr fragment of 30 amino acid residues from the photoaffinity-labeled enzyme. This CNBr fragment contains over 809% of the total label attached to prenyltransferase as a result of photoaffinity labeling. Several lines of evidence indicate that a number of residues in this CNBr fragment have been modified. First, Edman degradation of this labeled peptide demonstrates that at least 16 of the 30 amino acids have been modified by the photoaffinity reagent. The two most extensively modified amino acids are a specific arginine and alanine. Second, two-dimensional chromatography of Pronase digestions of the labeled CNBr fragment indicates that at least 11 different products resulted from photoaffinity labeling. Third, peptide maps of a trypsin digest of this CNBr fragment show that the attached affinity label is distributed among at least three of the resulting products of tryptic hydrolysis. Finally, comparison of amino acid analysis of this CNBr fragment with that of its counterpart isolated from native enzyme is consistent with the modification of a number of amino acids rather than a few y the photoaffinity labeling process.

Affinity Labels↗

PubMed1995

m-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: synthesis and application.

A novel photoaffinity label, m-acetylanilido-GTP (m-AcAGTP), was synthesized and used to identify GTP-binding proteins (G-proteins). This GTP analogue is easily prepared and can be used for photoaffinity labelling of G-proteins without chromatographic purification. In the presence of the beta-adrenergic agonist isoprenaline, it activates turkey erythrocyte adenylate cyclase. This activation persists even when the beta-adrenergic receptor is subsequently blocked by antagonist, indicating that the GTP analogue is resistant to hydrolysis. The apparent Ka for activation of turkey erythrocyte adenylate cyclase by m-AcAGTP was found to be 0.21 microM, a value similar to that for guanosine 5'-[beta,gamma-imido]triphosphate. m-AcAGTP also effectively inhibited the light-dependent GTPase of Musca fly eye membranes. Photoaffinity labelling of fly eye membranes with [alpha-32P]m-AcAGTP, followed by immunoprecipitation of G-protein Gq, identified a labelled protein band with the mobility of a 41.5 kDa protein on SDS/PAGE. Labelling of this protein was enhanced 9-fold in blue over red illuminated membranes, containing metarhodopsin and rhodopsin respectively. Labelling of alpha-subunits of heterotrimeric G-proteins was also demonstrated in turkey erythrocyte membranes. The ease of preparation of m-AcAGTP and the chemical properties of the photoreactive acetophenone make this affinity label an important new tool in studies of cellular phenomena mediated by guanine nucleotide-binding proteins.

Adenylyl Cyclases↗

PubMed1995

Identification and partial characterization of receptors for allatostatins in brain and corpora allata of the cockroach Diploptera punctata using a binding assay and photoaffinity labeling.

We have developed both an in vitro binding assay and a photoaffinity labeling assay to demonstrate and partially characterize putative receptors for allatostatins in brain and in corpora allata of Diploptera punctata. Isolated brain membranes were photoaffinity labeled with 125I-RYBPA (photoaffinity analogue of dip-allatostatin 5). Following labeling with 125I-RYBPA, SDS-PAGE and autoradiography revealed the presence of a putative receptor (37 kDa) for dip-allatostatin 5 and dip-allatostatin 7. Specific labeling was demonstrated by dose-dependent competition with either dip-allatostatin 5 or dip-allatostatin 7. The in vitro binding assay indicated that the receptor for dip-allatostatin 5 had a Kd of (9.0 +/- 0.9).10(-10) M and Bmax of 2.2 +/- 0.3 pmol/mg membrane protein. For dip-allatostatin 7, two Kd values of (1.5 +/- 0.1).10(-9) M and (3.8 +/- 0.3).10(-9) M were obtained, with Bmax values of 7.2 +/- 0.7 pmol/mg membrane protein and 11.4 +/- 1.0 pmol/mg membrane protein respectively. This indicates that there were probably two putative receptor sites for dip-allatostatin 7 although only one band was observable following photoaffinity labeling. Binding was saturable, specific and reversible. Using the in vitro binding assay, the Kd of the putative receptor in CA for dip-allatostatin 7 was shown to be (7.2 +/- 0.9).10(-10) M.

Affinity Labels↗

PubMed1996

HLA photoaffinity labeling reveals overlapping binding of homologous melanoma-associated gene peptides by HLA-A1, HLA-A29, and HLA-B44.

Melanoma-associated genes (MAGEs) encode tumor-specific antigens that can be recognized by CD8+ cytotoxic T lymphocytes. To investigate the interaction of the HLA-A1-restricted MAGE-1 peptide 161-169 (EADPT-GHSY) with HLA class I molecules, photoreactive derivatives were prepared by single amino acid substitution with N beta-[iodo-4-azidosalicyloyl]-L-2,3-diaminopropionic acid. These derivatives were tested for their ability to bind to, and to photoaffinity-label, HLA-A1 on C1R.A1 cells. Only the derivatives containing the photoreactive amino acid in position 1 or 7 fulfilled both criteria. Testing the former derivative on 14 lymphoid cell lines expressing over 44 different HLA class I molecules indicated that it efficiently photoaffinity-labeled not only HLA-A1, but possibility also HLA-A29 and HLA-B44. MAGE peptide binding by HLA-A29 and HLA-B44 was confirmed by photoaffinity labeling with photoreactive MAGE-3 peptide derivatives on C1R.A29 and C1R.B44 cells, respectively. The different photoaffinity labeling systems were used to access the ability of the homologous peptides derived from MAGE-1, -2, -3, -4a, -4b, -6, and -12 to bind to HLA-A1, HLA-A29, and HLA-B44. All but the MAGE-2 and MAGE-12 nonapeptides efficiently inhibited photoaffinity labeling of HLA-A1, which is in agreement with the known HLA-A1 peptide-binding motif (acidic residue in P3 and C-terminal tyrosine). In contrast, photoaffinity labeling of HLA-A29 was efficiently inhibited by these as well as by the MAGE-3 and MAGE-6 nonapeptides. Finally, the HLA-B44 photoaffinity labeling, unlike the HLA-A1 and HLA-A29 labeling, was inhibited more efficiently by the corresponding MAGE decapeptides, which is consistent with the reported HLA-B44 peptide-binding motif (glutamic acid in P2, and C-terminal tyrosine or phenylalanine). The overlapping binding of homologous MAGE peptides by HLA-A1, A29, and B44 is based on different binding principles and may have implications for immunotherapy of MAGE-positive tumors.

Affinity Labels↗

PubMed1997

Selective photoaffinity labelling of one mitochondrial protein in living cells of Saccharomyces cerevisiae with the fluorescent probe APMC. Identification of the target protein as subunit I of cytochrome c oxidase.

The lipophilic, cationic fluorochrome azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living yeast cells (Saccharomyces cerevisiae WT X 2180). It contains a photosensitive diazirine ring and is suitable for photoaffinity labelling. By combining photoaffinity labelling, micro-gel electrophoresis (SDS-PAGE), and detection of the APMC fluorescence with a microfluorimeter, we established a highly sensitive procedure for determining the apparent molecular weight of the APMC-labelled proteins in yeast cells. On vital staining at 0.1 microM APMC for 30 min, only one mitochondrial protein with an apparent molecular weight of 40 kDa is labelled with high intensity. At increased dye concentrations proteins of 47 and 49 kDa are labelled too, however not until all binding sites of the 40 kDa protein are occupied. Obviously, the APMC cations have a pronounced affinity for this protein. It was shown by fractional centrifugation that the labelled 40 kDa protein is a constituent of the inner mitochondrial membrane. One driving force for the accumulation of the APMC cations is the trans-membrane potential (TMP) across the inner mitochondrial membrane. Consequently, uncouplers like dinitrophenol (DNP) and carbonylcyanidechlorophenyl-hydrazone (CCCP), ionophores (valinomycin, gramicidin), and inhibitors of the respiratory chain (myxothiazol, KCN), which decrease the TMP, also diminish the APMC accumulation and labelling. And conversely, drugs, which hyperpolarize the inner membrane (nigericin, atractyloside), favour APMC labelling. Another driving force of APMC accumulation is the dye's lipophilicity, which facilitates dye accumulation by hydrophobic interaction with the very lipophilic proteins of the inner mitochondrial membranes. This was shown by competitive double staining experiments. Thiamine strongly inhibits APMC labelling. Obviously, the transport of the APMC cations is facilitated by the thiamine carrier, and thiamine competes for the same binding sites, which are occupied by the dye cations. Chloramphenicol is an inhibitor of the mitochondrial protein synthesis without affecting the TMP. On preincubation, chloramphenicol completely quenches the signal of the 40 kDa protein. Therefore, this protein must be encoded on the mtDNA. The only 40 kDa protein with adequate properties is the subunit I of cytochrome c oxidase. Obviously, it is the preferred target of the APMC cations on photoaffinity labelling. This assignment agrees with the strong hydrophobicity of the labelled 40 kDa protein, which was tested with various detergents. It also agrees with the solvatochromism of the protein-bound APMC label, and finally with the paralellism of the labelled protein with cytochrome c oxidase on fractional ammonium sulfate precipitation.

Carbocyanines↗

PubMed1994

Larva lights: a decade of photoaffinity labeling with juvenile hormone analogues.

The introduction of photoaffinity labeling into the mode of action of insect hormones and pheromones started 12 yr ago with the photoaffinity labeling of juvenile hormone binding proteins (JHBPs) from cockroaches in the laboratory of the late John K. Koeppe. Applying this technique to Manduca sexta led ultimately to a three-laboratory collaborative project that has begun to dissect the molecular basis for JH transport, metabolism, and nuclear binding and gene activation in Lepidoptera. This review provides (1) a history of the first experiments; (2) an idea of the breadth of the technique in the arthropod classes Insecta, Crustacea, and Arachnida; and (3) evidence for the depth of the technique in unearthing key details about three different types of the molecular action of JH in M. sexta.

Affinity Labels↗

PubMed1986

Solubilization and separation of the human erythrocyte D-glucose transporter covalently and noncovalently photoaffinity-labeled with [3H]cytochalasin B.

The D-glucose transporter in the human erythrocyte membranes was photoaffinity-labeled with [3H]cytochalasin B and solubilized with n-octyl beta-D-glucopyranoside (octyl glucoside). [3H]Cytochalasin B-bound proteins were further isolated by using Sephadex G-50 chromatography. The amount of [3H]cytochalasin B associated with the membrane proteins was approximately 10% of the total radioactivity in the octyl glucoside extract. The solubilized photoaffinity-labeled D-glucose transporter was isolated and found to consist of two major peaks by DEAE-Sephacel chromatography. The radioactivity of peak II was considerably greater than that of peak I. The incorporation of [3H]cytochalasin B into both peaks was blocked by the presence of D-glucose during photolysis. With preparative NaDod-SO4/polyacrylamide gel electrophoresis, the radioactivity of peak I could be released, but that of peak II remained with the D-glucose transporter. These results indicate that [3H]cytochalasin B was covalently bound to the D-glucose transporter only in peak II and that peak II could be generated by the photoaffinity labeling of peak I. However, the D-glucose transport activity was associated only with peak I. These findings suggest that the anionic domain of the D-glucose transporter becomes exposed because of conformational changes of the protein as a result of covalent binding with [3H]cytochalasin B by photoaffinity labeling.

Affinity Labels↗

PubMed1987

Photoaffinity labeling of felodipine-binding proteins in vascular smooth muscle.

[3H]-Felodipine and high-intensity ultraviolet irradiation were used in the photoaffinity labeling of soluble proteins prepared from porcine mesenteric vascular smooth muscle. Irradiation of the soluble proteins in the presence of [3H]-felodipine resulted in the labeling of a protein with an apparent molecular weight of 62 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Labeling of the protein did not occur without ultraviolet irradiation. An [3H]-azido analog of felodipine was found to show less specificity than felodipine in its protein labeling when irradiated, since proteins with apparent molecular weights of 44, 29, and 14, as well as 62 kDa, were labeled. The photoaffinity labeling of the proteins were inhibited by excess of unlabeled felodipine.

Affinity Labels↗