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Michael J Martinelli - One of the best experts on this subject based on the ideXlab platform.

  • characterization by Photoaffinity Labelling of the human platelet thromboxane a2 prostaglandin h2 receptor evidence for n linked glycosylation
    European Journal of Pharmacology, 1992
    Co-Authors: Dale E. Mais, Timothy A True, Michael J Martinelli
    Abstract:

    Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) are potent proaggregatory and vasoconstrictor lipids acting through a receptor referred to as the TXA2/PGH2 receptor. The receptor was purified using a modification of a previously described method from human platelet membranes solubilized using the detergent (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and a combination of affinity chromatography and wheat germ lectin chromatography. This procedure resulted in a 1075 +/- 375-fold purification and a specific activity of 1.45 +/- 0.55 nmol/mg protein (n = 5). Repeating these chromatography steps on this partially purified receptor resulted in a preparation with a specific activity of 21 +/- 3 nmol/mg protein (n = 5). This represents the theoretical specific activity if one assumes a molecular weight of 50,000 for the receptor. The fold purification was 11,750 +/- 1250 based on crude membranes and an overall yield of 24%. To further the characterization of this receptor, we synthesized a new radioiodinated Photoaffinity probe, 7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4-azido-3-iodobenzenesulfonylamino++ + )- bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid (I-SAP-N3). [125I]l-SAP-N3 irreversibly incorporated into the purified receptor yielding a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography and indicated a molecular weight for the receptor of 50-51 kDa. The incorporation of the ligand could be inhibited by a variety of TXA2/PGH2 analogues. In addition, Photoaffinity Labelling was inhibited in a stereoselective manner as demonstrated by the pair of enantiomers (d)- and (l)-S145. Digestion of Photoaffinity labelled receptor with N-glycosidase F demonstrated the presence of at least two N-linked glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Charles H. Hocart - One of the best experts on this subject based on the ideXlab platform.

  • the synthesis of h labelled 8 azido n benzyladenine and related compounds for Photoaffinity Labelling of cytokinin binding proteins
    Molecules, 2019
    Co-Authors: David S. Letham, Charles H. Hocart, Xue-dong Zhang
    Abstract:

    The biology of the group of plant hormones termed cytokinins is reviewed to reveal areas where further studies of cytokinin-binding proteins could be significant. Such areas include: inhibition of human tumour cell growth by cytokinin ribosides, the role of cytokinins in the development of diverse micro-organisms including the cyanobacteria and Mycobacterium tuberculosis, the very rapid responses of plant cells to exogenous cytokinins, and other aspects of cytokinin plant biology. Photoaffinity Labelling (PAL) coupled to the recent advances in HPLC of proteins and mass spectral analysis and sequencing of proteins, may have relevance to these areas. To facilitate PAL, we present experimental details for two methods for synthesis of 8-azido-N⁶-benzyladenine, which has the azido affinity group in the preferred position of the purine ring. Synthesis from [2-³H]adenosine yielded the above-mentioned PAL reagent with ³H in the purine ring and also gave labelled 9-riboside and 8-azido-N⁶,9-dibenzyladenine. 8-Azido-N⁶-benzyladenine was also prepared from 6,8-dichloropurine by a facile synthesis, which would allow a label to be sited in the benzyl group where substituents can also be introduced to vary cytokinin activity. The use of inactive cytokinin analogues in assessing the significance of PAL is discussed.

  • The Synthesis of 3H-Labelled 8-Azido-N6-Benzyladenine and Related Compounds for Photoaffinity Labelling of Cytokinin-Binding Proteins
    MDPI AG, 2019
    Co-Authors: David S. Letham, Xue-dong Zhang, Charles H. Hocart
    Abstract:

    The biology of the group of plant hormones termed cytokinins is reviewed to reveal areas where further studies of cytokinin-binding proteins could be significant. Such areas include: inhibition of human tumour cell growth by cytokinin ribosides, the role of cytokinins in the development of diverse micro-organisms including the cyanobacteria and Mycobacterium tuberculosis, the very rapid responses of plant cells to exogenous cytokinins, and other aspects of cytokinin plant biology. Photoaffinity Labelling (PAL) coupled to the recent advances in HPLC of proteins and mass spectral analysis and sequencing of proteins, may have relevance to these areas. To facilitate PAL, we present experimental details for two methods for synthesis of 8-azido-N6-benzyladenine, which has the azido affinity group in the preferred position of the purine ring. Synthesis from [2-3H]adenosine yielded the above-mentioned PAL reagent with 3H in the purine ring and also gave labelled 9-riboside and 8-azido-N6,9-dibenzyladenine. 8-Azido-N6-benzyladenine was also prepared from 6,8-dichloropurine by a facile synthesis, which would allow a label to be sited in the benzyl group where substituents can also be introduced to vary cytokinin activity. The use of inactive cytokinin analogues in assessing the significance of PAL is discussed

H W Zimmermann - One of the best experts on this subject based on the ideXlab platform.

  • 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
    Journal of Photochemistry and Photobiology B-biology, 1997
    Co-Authors: H Haassmannle, H W Zimmermann
    Abstract:

    Abstract The lipophilic, cationic fluorochrome azopnetylmethylindocarbocyanine (APMC) specifically stains the mitochondria in a 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 μM 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-hydrzone (CCCP), ionophores (valinomycin, gramiciding), 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, atractloside), 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 o 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 lable, and finally with the paralellism of the labelled protein with cytochrome c oxidase on fractional ammonium sulfate precipitation.

  • influence of trans membrane potential and of hydrophobic interactions on dye accumulation in mitochondria of living cells Photoaffinity Labelling of mitochondrial proteins action of potential dissipating drugs and competitive staining
    Histochemistry and Cell Biology, 1994
    Co-Authors: K Schneider, A Naujok, H W Zimmermann
    Abstract:

    The lipophilic cationic fluorescent dye azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living cells. The dye contains a photosensitive diazirine ring and is suitable for Photoaffinity Labelling of mitochondrial proteins. By a combination of Photoaffinity Labelling of cell cultures of mouse fibroblasts (LM) with APMC, lysis of the labelled cells, subsequent micro-gel electrophoresis and detection of the fluorescence of the labelled proteins in the gel lanes with a sensitive microfluorimeter, we determined the number, apparent molecular masses, and relative intensity of the labelled proteins. In LM cells, three proteins with apparent molecular masses of 31, 40, and 74 kDa were labelled with high intensity, and proteins of 28, 29, 44, 48, 49, 66, and 105 kDa with low intensity. Two effects mainly determine the binding of lipophilic dye cations to mitochondrial proteins in living cells: (1) interaction of the trans-membrane potential of the inner mitochondrial membrane with the dye cations; and (2) hydrophobic interactions between the strongly lipophilic proteins of the inner membrane and the lipophilic dye molecules. Preincubation of the cell cultures with drugs that dissipate the trans-membrane potential, such as valinomycin, 2,4-dinitrophenol (DNP) and 3-chlorcarbonylcyanidephenylhydrazon (CCCP), strongly reduces or even prevents APMC Labelling of mitochondrial proteins. The influence of hydrophobic interactions was investigated by competitive staining experiments using dyes with very different lipophilic properties. The lipophilicity of the dyes was characterized by their R m values in reversed phase thin-layer chromatography. Prestaining with an excess of strongly lipophilic cationic acridine and phenanthridine dyes, such as pentyl acridinium orange chloride (PAO), nonyl acridinium orange chloride (NAO) and tetramethylpropidium chloride (MP), respectively, completely prevents protein Labelling with APMC. Obviously, the dyes occupy the same mitochondrial binding sites as APMC. At equal concentrations the intensity of the 40-kDa signal is strongly reduced, whereas the 31-kDa and 74-kDa signals are unaffected. Using phenanthridine dyes with lower lipophilicity, namely propidium chloride (P), M, and N reduces the peak of the 40-kDa protein in APMC Labelling, indicating that the 40-kDa protein preferentially binds lipophilic dye cations.

  • Photoaffinity Labelling with fluorescence detection dye accumulation at four mitochondrial proteins in hela and lm cells
    Histochemistry and Cell Biology, 1994
    Co-Authors: K Schneider, H W Zimmermann
    Abstract:

    A micromethod was developed for investigating the interactions between fluorescent dyes and cellular proteins. The lipophilic cationic dye APMC (azopentylmethylcarbocyanine) contains a photosensitive diazirine ring and is suitable for Photoaffinity Labelling. By combining Photoaffinity Labelling of cultured cells, micro-gel electrophoresis and detection of the fluorescence with a microfluorimeter, we established a highly sensitive and rapid procedure to identify APMC labelled proteins. Cells which had been incubated for 10 min with 10−8M APMC could be analysed for APMC binding without difficulty. Under our experimental conditions this corresponds to about 0.2 nmol APMC per mg protein. The lipophilic APMC specifically stains the mitochondria in living HeLa and LM cells. The fluorescing mitochondria can be easily detected under a fluorescence microscope. By Photoaffinity Labelling we were able to show that at low dye concentrations APMC preferentially marks four proteins with apparent molecular masses of 31, 40, 66, and 74 kDa. In order to establish that these are mitochondrial proteins, we isolated and analysed the mitochondria from incubated HeLa and LM cells; again, the same four proteins were detected. They are most probably proteins of the inner mitochondrial membranes, which accumulate the lipophilic APMC cations.

Geoffrey D Holman - One of the best experts on this subject based on the ideXlab platform.

  • Synthesis of biotinylated bis(D-glucose) derivatives for glucose transporter Photoaffinity Labelling.
    Carbohydrate Research, 2001
    Co-Authors: Makoto Hashimoto, Yasumaru Hatanaka, Jing Yang, Jaswant Dhesi, Geoffrey D Holman
    Abstract:

    New diazirine based bis-glucose derivatives for tagging glucose transporters have been synthesised. These included two biotinylated compounds linked either by an aminocaproate or by a cleavable dithiol link. These compounds have been derivatised via a key skeleton compound that can be easily used for introduction of additional tags. Studies on the erythrocyte glucose transporter (GLUT1) and the insulin-stimulated adipose cell transporter (GLUT4) have revealed the biotinylated photoreactive bis-glucose compounds are effective Labelling reagents.

J Baker - One of the best experts on this subject based on the ideXlab platform.

  • radioligand binding and Photoaffinity Labelling studies show a direct interaction of phenothiazines at 5 ht3 receptors
    Neuropharmacology, 1997
    Co-Authors: Sarah C R Lummis, J Baker
    Abstract:

    Abstract The effects of a range of phenothiazines were examined on 5-hydroxytryptamine3 (5-HT3) receptors in membranes from N1E-115 neuroblastoma cells using radioligand binding. Chlorpromazine, fluphenazine, perphenazine, trifluoperazine and prochlorperazine inhibited specific binding of both the 5-HT3 receptor antagonist [3H]GR65630 and agonist [3H]meta-chlorophenylbiguanide (mCPBG), with Ki values ranging from 0.4 to 3.9 μM. The mode of action of chlorpromazine was further examined using Photoaffinity Labelling in the presence and absence of 5-HT. Saturation radioligand binding data with both [3H]GR65630 and [3H]mCPBG showed that Photoaffinity Labelling with chlorpromazine (1 μM) caused a decrease in the maximum number of binding sites observed (35% and 28% for agonist and antagonist, respectively). This decrease was not observed when the membranes were incubated in the presence of 5-HT. The results demonstrate a direct interaction of a range of phenothiazines at the 5-HT3 receptor binding site. © 1997 Elsevier Science Ltd.