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PubMed1984

Purification of the photoaffinity-labeled glucagon receptor by gel electrophoretic methods.

Rat liver plasma membrane glucagon receptor has been purified with a yield of 0.01% to an estimated homogeneity of 32-60%, using a 2-stage electrophoretic procedure. SDS-solubilized membrane proteins labeled by the photoaffinity-agent, Ne-4-azidophenylamidinoglucagon (APA-glucagon), were separated by polyacrylamide gel electrophoresis in SDS-containing buffers. Gel slices corresponding to the molecular weight of the receptor were excised, electrophoretically extracted and concentrated. The concentrate was subjected to isoelectric focusing on Sephadex to yield a purified product in which the photoaffinity-labeled receptor, with a molecular weight of 56K and a pI' of 5.9, is the sole major component.

Affinity Labels↗

PubMed1992

Photoaffinity labeling of the organic cation/H+ exchanger in renal brush border membrane vesicles.

The brush border membrane of the proximal tubule contains two efflux pathways for organic cations from the cell to the tubular fluid: a P-glycoprotein and an organic cation/H+ exchanger. There is evidence that they transport many of the same substrates. Their structural relatedness is unknown and is the subject of this report. The experimental approach was to identify the exchanger with photoaffinity labeling reagents. The rationale was that if the P-glycoprotein and the organic cation/H+ exchanger transport many of the same substrates, then they might be photoaffinity labeled by the same reagents. [125I]Iodoarylazidoprazosin and [3H]azidopine are two reagents, which have been used, to photoaffinity label the P-glycoprotein. We found that several polypeptides were photolabeled in a time- and concentration-dependent manner. The photoincorporation into only two of these polypeptides (41 and 28 kDa) was blocked extensively by the presence of known substrates for the exchanger. The photoaffinity labeling of only the 41-kDa polypeptide was affected by treatment with the chemical reagents, N-ethylmaleimide and dithiothreitol, which are known to affect the exchanger reaction. The findings are consistent with the interpretation that a 41-kDa polypeptide is, or is a component of, the exchanger.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

The gamma 2 subunit is an integral component of the gamma-aminobutyric acidA receptor but the alpha 1 polypeptide is the principal site of the agonist benzodiazepine photoaffinity labeling reaction.

Polyclonal antibodies were raised to a synthetic peptide whose amino acid sequence was derived from the novel gamma-aminobutyric acidA (GABAA) receptor subunit, gamma 2. These anti-gamma 2 1-15 Cys antibodies reacted specifically with the GABAA receptor purified from adult bovine cerebral cortex in an enzyme-linked immunosorbent assay. Anti-gamma 2 1-15 Cys antibodies specifically immunoprecipitated [3H]flunitrazepam photoaffinity-labeled native receptor in parallel with anti-alpha 1 324-341 antibodies. Immunoprecipitation of sodium dodecyl sulphate (SDS) denatured photoaffinity-labeled receptor by anti-gamma 2 1-15 Cys antibodies, however, resulted in a significant decrease in the maximum percentage of radioactivity immunoprecipitated compared to that by anti-alpha 1 324-341 antibodies. In immunoblots, anti-gamma 2 1-15 Cys antibodies reacted with a broad band in the molecular weight range Mr 43,000-49,000 which was distinct from that recognized by anti-alpha 1 324-341 antibodies. The anti-alpha 1 324-341 immunoreactive band was the main subunit irreversibly photoaffinity labeled by [3H]flunitrazepam, i.e. Mr 53,000. These results demonstrate for the first time that the gamma 2 subunit is an integral component of the GABAA receptor but it is the alpha 1 subunit that is the principal site of the agonist benzodiazepine photoaffinity labeling reaction. It supports a role of both the alpha 1 and gamma 2 polypeptides in the formation of the central benzodiazepine binding site within a GABAA receptor oligomer.

Affinity Labels↗

PubMed1983

Photoaffinity labeling: theory and practice.

The potential for generating a reactive reagent in situ by photoexcitation and for labeling sites not containing reactive nucleophilic residues provides photoaffinity labeling with a special advantage in the study of ligand-binding sites. There are, however, problems in the application of the photoaffinity technique. A number of concerns about this approach are examined by using as a background studies with arylazido-beta-alanyl nucleotide probes developed in this laboratory. It is concluded that photoaffinity labeling is likely to emerge as one of the major techniques for elucidating structure-function relationships within biological membranes. In the future the design of photoprobes with shorter activation half-lives may make it possible to investigate time-dependent (i.e., conformational) changes occurring at the active site of enzymes and within complex ligand-binding sites.

Adenosine Triphosphate↗

PubMed2000

Synthesis and preliminary analysis of a P-glycoprotein-specific [3H]-benzophenone photoaffinity label based on (-)-stipiamide.

A benzophenone photoaffinity label 9 based on the polyene natural product (-)-stipiamide has been constructed using a diaminoethane spacer and the radioactive agent [3H]-BZDC (N-succinimidyl p-benzoyl-(2,3-3H)-dehydrocinnamate). Photoaffinity experiments show specific binding to human P-glycoprotein (Pgp) in the presence of cis-flupentixol but not with cyclosporin A.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Evaluation of comparative cytochrome P450 2B4 model by photoaffinity labeling.

A homology model of rabbit CYP 2B4 was constructed on the basis of the crystallographic structure of truncated mammalian CYP 2C5/3 and bacterial soluble CYPs. To validate the CYP 2B4 homology model photoaffinity labeling was employed. Three probes (I-III) containing a photo-labile azido-group and an amino-group on opposite ends of the molecule were designed for photoaffinity labeling of the CYP 2B4 in increasing distance from the heme iron. Spectroscopic data proved probes I (the shortest) and II (a middle sized) to be coordinated with the heme iron via their amino-groups in the enzyme active center while the probe III (the longest) was not bound in this way. This binding orientation of probes I and II is in accordance with the model predicting ion-pairing of the negatively charged side chain of CYP 2B4 Asp 105 and a positively charged nitrogen located in an appropriate position in structures of probes I and II, only. The lack of heme binding of the probe III is clear from its docking into the CYP 2B4 model since no Asp 105 ion-pairing is possible. The target of photoactivated probe II, Arg 197, in a distance of about 16.5 A from the heme iron, exactly matches the position of that amino acid residue, predicted from the CYP 2B4 homology model. Moreover, using this technique, a substrate access channel has been identified. To assess the predicted substrate-binding pocket, an interaction of a specific CYP 2B4 substrate, diamantane, was examined. In "silico" docking revealed strong binding of diamantane in an orientation allowing experimentally observed C4-hydroxylation. Our homology model of CYP 2B4 is thus consistent with experimental metabolic and photoaffinity labeling data.

Amino Acid Sequence↗

PubMed1995

Photoaffinity labeling of retinoic acid-binding proteins.

Retinoid-binding proteins are essential mediators of vitamin A function in vertebrate organisms. They solubilize and stabilize retinoids, and they direct the intercellular and intracellular trafficking, transport, and metabolic function of vitamin A compounds in vision and in growth and development. Although many soluble retinoid-binding proteins and receptors have been purified and extensively characterized, relatively few membrane-associated enzymes and other proteins that interact with retinoids have been isolated and studied, due primarily to their inherent instabilities during purification. In an effort to identify and purify previously uncharacterized retinoid-binding proteins, it is shown that radioactively labeled all-trans-retinoic acid can be used as a photoaffinity labeling reagent to specifically tag two known retinoic acid-binding proteins, cellular retinoic acid-binding protein and albumin, in complex mixtures of cytosolic proteins. Additionally, a number of other soluble and membrane-associated proteins that bind all-trans-[11,12-3H]retinoic acid with high specificity are labeled utilizing the same photoaffinity techniques. Most of these labeled proteins have molecular weights that do not correspond to any known retinoid-binding proteins. Thus, photoaffinity labeling with all-trans-retinoic acid and related photoactivatable retinoids is a method that should prove extremely useful in the identification and purification of novel soluble and membrane-associated retinoid-binding proteins from ocular and nonocular tissues.

Affinity Labels↗

PubMed1999

Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling.

The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver.

Adult↗

PubMed1996

Protein folding within and protein transport into mammalian microsomes are differentially affected by photoaffinity labeling of microsomes with 8-azido-ATP.

Transport of presecretory proteins into mammalian microsomes involves a microsomal protein which is sensitive to photoaffinity labeling with 8-azido-ATP. Typically, protein folding within the lumen of the endoplasmic reticulum of mammalian cells depends on ATP and the member of the Hsp70 protein family, BiP. Here we addressed the question of whether protein transport into and folding within microsomes are differentially affected by photoaffinity labeling of microsomes with 8-azido-ATP. Folding of heterodimeric luciferase to the native state was more azido-ATP-sensitive compared to transport of the precursors of the two subunits. Therefore, we conclude that the microsomal protein which is responsible for the ATP-dependence of protein folding in the endoplasmic reticulum is sensitive to photoaffinity labeling with 8-azido-ATP and that this microsomal protein is distinct from the microsomal ATP-binding protein which is involved in protein transport.

Adenosine Triphosphate↗

PubMed1983

Photoaffinity-labelling of the glycine receptor of rat spinal cord.

The irreversible incorporation upon ultraviolet illumination of the glycine receptor antagonist, [3H]strychnine, into synaptic membrane fractions of rat spinal cord has been investigated. The specificity of this photoaffinity-labelling reaction for the glycine receptor was demonstrated by the following results: (a) the Kd value (9.7 nM) of the glycine-displaceable irreversible incorporation of [3H]strychnine was similar to the previously reported Kd of [3H]strychnine binding to the glycine receptor; (b) pre-illumination of the membranes with unlabelled strychnine led to a corresponding reduction in the number, but not the affinity, of reversible glycine-displaceable [3H]strychnine binding sites; (c) the ultraviolet light-induced incorporation into the membranes of [3H]strychnine was inhibited by different glycine receptor agonists; other neurotransmitter substances had little or no effect. Also, [3H]strychnine alone was shown to be stable upon illumination with ultraviolet light; this suggests that photocrosslinking of [3H]strychnine may require energy transfer from specific groups of its high-affinity receptor binding site. Upon sodium dodecyl sulphate/polyacrylamide gel electrophoresis a single labelled polypeptide with a relative molecular mass of 48000 was revealed from spinal cord membranes photoaffinity-labelled with [3H]strychnine. Spinal cord membranes photoaffinity-labelled with the gamma-aminobutyric acid receptor ligand [3H]flunitrazepam, however, gave a single polypeptide with a relative molecular mass of 5- 0000. Treatment of membranes, labelled with [3H]strychnine, by endoglycosidase H did not alter the relative molecular mass of the 48000-Mr labelled polypeptide. Trypsin treatment, on the other hand, successively produced major fragments of relative molecular masses of 42000 and 37000. Also, even after extensive treatment with trypsin or chymotrypsin, greater than or equal to 90% of the radioactivity incorporated into the labelled membranes remained membrane-associated. It is concluded that the strychnine binding site of the glycine receptor is located on a protease-inaccessible, i.e. probably hydrophobic domain of the 48000-Mr subunit.

Affinity Labels↗

PubMed1985

Photoaffinity labelling of androgen receptors with 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one.

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in calf uterus and rat prostate. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of 98 kD. In rat prostate cytosol an androgen receptor with a molecular mass of 46 kD could be photoaffinity labelled with R1881. The photoaffinity labelling procedure described here provides a method for studying the hormone binding domain of androgen receptors in partial purified preparations.

Affinity Labels↗

PubMed1986

Photoaffinity labeling of the gonadotropin receptor with native, asialo, and deglycosylated choriogonadotropin.

Human choriogonadotropin (hCG) is a heterodimeric hormone composed of an alpha and a beta subunit. hCG and its asialo (ashCG) and deglycosylated (dghCG) forms vary in their ability to stimulate hormone responsive adenylate cyclase. ashCG is a partial agonist, and dghCG is an antagonist. Photoactivatable moieties were coupled to hCG, ashCG, and dghCG, and the derivatives were radioiodinated. Competitive binding studies indicate that all of the derivatives had a similar affinity for the gonadotropin receptor on porcine granulosa cell membranes. Radiolabeled derivatives were used to photoaffinity label the gonadotropin receptor. Radiolabeled complexes were separated by NaDodSO4/PAGE. All of the derivatives produced similar autoradiographic patterns, except that dghCG produced an additional 48-kDa complex. To investigate the structure of the complexes further, peptide mapping of proteolytic digests was used. All, except for the 48-kDa complex, generated similar peptide maps indicating a relationship between those complexes in which the smaller components are part of the larger. The 48-kDa complex contained both subunits of 40-kDa dghCG. Therefore, this complex is expected to contain an additional component of 8 kDa. The complex was generated whether the hormone-receptor complex was photoaffinity labeled on cells, on isolated membranes, or after solubilizing in detergent. Formation was blocked by excess hCG and did not occur in the absence of UV irradiation. We conclude that the hCG derivatives are able to photoaffinity label the hCG receptor but that the dghCG derivative can photoaffinity label an additional component that was not observed when derivatives of hCG or ashCG were used to label the receptor.

Adenylyl Cyclases↗

PubMed1998

Photoaffinity labeling of PKC isozymes by phorbol ester derivatives.

Photoaffinity probes PPDA and PPTD, which have diazoacetyl and trifluorodiazopropionyl group at C-13 position in phorbol, respectively, were synthesized. Photoaffinity labeling of protein kinase C isozymes by both the probes resulted in specific cross-linking.

Cross-Linking Reagents↗

PubMed1992

Effects of GTP analogues and activation of endogenous protein kinases on photoaffinity labeling with [3H](+)PN200-110 of crude membranes from rat heart and brain.

The effects of GTP analogues and conditions in which various endogenous protein kinases were activated on photoaffinity labeling with [3H](+)PN200-110 (PN) of crude membranes from rat cardiac muscle and whole brain were investigated. Photoaffinity labeling with 20 nM [3H](+)PN of these crude membranes was decreased by 100 microM GTP-gamma-S, but not by 100 microM GTP or 100 microM GDP-beta-S. Similar results were obtained on the effects of GTP and its analogues on the specific binding of 20 nM [3H](+)PN to these crude membranes under the same conditions. Activation of endogenous protein kinases in these crude membranes did not influence the photoaffinity labeling with [3H](+)PN. These results suggested the binding sites, or DPH-sensitive, or L-type, calcium channels in curde membranes from rat cardiac muscle and whole brain are directly or indirectly modulated by endogenous GTP-binding protein, but not by various endogenous protein kinases in these crude membranes.

Affinity Labels↗

PubMed1998

Identification of binding domains of pituitary adenylate cyclase activating polypeptide (PACAP) for its type 1 receptor by photoaffinity labeling.

Structure-function studies and photoaffinity labeling experiments were performed to identify residues and domains of PACAP involved in the interaction with PACAP receptors. For this purpose, a series of photoreactive analogues of PACAP(1-27) containing a photoreactive benzophenone (BP) residue in different peptide structural domains were utilized to analyze the interaction of PACAP(1-27) with pig PACAP type 1 receptors. Five PACAP derivatives were created with a photoreactive amino acid in the following peptide domains: either the disordered N-terminal or the helical C-terminal domain or a short loop region within the C-terminal helical domain of the peptide. Their receptor binding properties and efficiencies were tested on pig brain PACAP receptors. The results indicate the importance of the helical C-terminal domain of PACAP(1-27) for receptor binding affinity. Monoiodination of the photoreactive analogues did not change their binding affinities. Experiments with pig brain membranes demonstrated that the 125I-labeled photoreactive analogues specifically label a protein band of M(r) 66,000. The efficiency of photoreactive labeling differed for the various analogues. These findings suggest that Tyr22 and Lys15 in PACAP (1-27) are located in or close to the hormone binding site of the PACAP type 1 receptor. The results provide evidence that the alpha-helical C-terminal region of PACAP is directly involved in receptor binding.

Amino Acid Sequence↗

PubMed1993

Catechol O-methyltransferase pharmacogenetics: photoaffinity labelling and western blot analysis of human liver samples.

The level of catechol O-methyltransferase (COMT) activity and COMT thermal stability in human tissue are controlled by a common genetic polymorphism. We studied individual hepatic biopsy samples shown previously to have phenotypically high, low or intermediate COMT activities and thermal stabilities to test the hypothesis that the molecular mass (M(r)) and/or isoelectric point (pI) of the enzyme might differ in tissue from subjects with different presumed genotypes for the COMT genetic polymorphism. COMT was partially purified from each hepatic tissue sample by sequential ion exchange and gel filtration chromatography, and photoaffinity labelling was performed with [3H-methyl]-S-adenosyl-L-methionine ([3H-methyl]-Ado-Met), the methyl donor for the COMT enzymatic reaction. Two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) analysis of individual samples consistently showed the presence of three [3H-methyl]-Ado-Met photoaffinity labelled proteins with pI values of 5.4, 5.5 and 5.7, all three of which had M(r) values of approximately 27.1 kDa. The same pattern was observed in all samples irrespective of COMT phenotype. Western blot analysis of 2-D SDS-PAGE gels performed with rabbit polyclonal antibodies to partially purified human kidney COMT showed a pattern similar to that found during photoaffinity labelling. Once again, the same pattern was found in all samples irrespective of COMT phenotype. Therefore, neither photoaffinity labelling nor Western blot analysis revealed differences in either M(r) or pI of cytoplasmic COMT in hepatic tissue from subjects selected on the basis of different phenotypic expression of the COMT genetic polymorphism.

Affinity Labels↗

PubMed1999

Photoaffinity labeling and mass spectrometry identify ribosomal protein S3 as a potential target for hybrid polar cytodifferentiation agents.

The ability of a novel class of hybrid polar compounds (HPCs) to induce differentiation and consequent cessation of proliferation of transformed cells has led to their development as potential chemotherapeutic agents in the treatment of cancer. Suberoylanilide hydroxamic acid (SAHA) is a prototype of a family of hydroxamic acid based compounds (SAHA-like HPCs) that can, at micromolar concentrations, induce a variety of transformed cell lines to differentiate. The mechanism of action of the HPCs is not entirely understood. Searching for a cellular target of the SAHA-like HPCs, we synthesized a photoaffinity labeling reagent structurally based on SAHA, and probed for SAHA-binding proteins in murine erythroleukemia (MEL) cells. Photoaffinity labeling in cell free extracts identified a 32-kDa protein (p32) that was specifically labeled by the photoaffinity reagent. Cell fractionation assays localized p32 to the P100 fraction. p32 was partially purified and identified by mass spectrometry as the 40 S ribosomal protein S3. Expression of epitope-tagged S3 in bacterial lysates followed by photoaffinity labeling confirmed its specific labeling. Identification of a cytodifferentiation agent target may shed light on the mechanism by which the SAHA-like HPCs exert their antitumor effects.

Affinity Labels↗

PubMed1985

8-Azidoflavins as photoaffinity labels for flavoproteins.

8-Azidoflavins have been synthesized and their potential as photoaffinity labels for flavoproteins has been explored. They are very photolabile, and in aqueous media they react with solvent to yield 8-aminoflavins and 8-hydroxlaminoflavins as the main products. They fulfill the criteria expected of a good photoaffinity label, since they bind stoichiometrically at the flavin-binding site of flavoproteins, thus minimizing problems of nonspecific labeling. Second, they absorb strongly in the visible, so that the reactive nitrene can be generated without short wavelength light, minimizing the possibility of light-induced damage of the protein. Third, in the absence of light, 8-N3-flavins are stable, permitting a study of their binding to apoproteins. 8-Azidoflavins have been bound to hen egg white riboflavin-binding protein, Megasphera elsdenii flavodoxin, yeast Old Yellow Enzyme, Aspergillus niger, glucose oxidase, and pig kidney D-amino acid oxidase, and the effect of exposure to visible light has been determined. Only small extents of covalent attachment of the flavin to the protein were found with flavodoxin, D-amino acid oxidase, and Old Yellow Enzyme; much more extensive labeling was obtained with glucose oxidase and riboflavin-binding protein. In addition to their photoreactivity, 8-azidoflavins have been found to be converted to 8-aminoflavins by reaction with sulfite or upon reduction. Similar reactions occur with 8-hydroxylamino-, 8-(O-methyl)hydroxylamino-, and 8-hydrazinoflavins, which serve as models for possible flavin-protein covalent linkages which could be formed in the photolabeling procedure. Some of the properties of these flavins, which were obtained by reaction of 8-F-flavin with the corresponding nucleophiles, are also described.

Affinity Labels↗