Статті в журналах: "Proteins – Affinity labeling"

1

SWEET, FREDERICK, and GARY L. MURDOCK. "Affinity Labeling of Hormone-Specific Proteins*." Endocrine Reviews 8, no. 2 (May 1987): 154–84. http://dx.doi.org/10.1210/edrv-8-2-154.

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2

Vinkenborg, Jan L., Günter Mayer, and Michael Famulok. "Aptamer-Based Affinity Labeling of Proteins." Angewandte Chemie International Edition 51, no. 36 (August 2, 2012): 9176–80. http://dx.doi.org/10.1002/anie.201204174.

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3

Löw, Andreas, Heinz G. Faulhammer, and Mathias Sprinzl. "Affinity labeling of GTP-binding proteins in cellular extracts." FEBS Letters 303, no. 1 (May 25, 1992): 64–68. http://dx.doi.org/10.1016/0014-5793(92)80478-y.

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4

Song, Yinan, Feng Xiong, Jianzhao Peng, Yi Man Eva Fung, Yiran Huang, and Xiaoyu Li. "Introducing aldehyde functionality to proteins using ligand-directed affinity labeling." Chemical Communications 56, no. 45 (2020): 6134–37. http://dx.doi.org/10.1039/d0cc01982h.

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5

Maldonado, H. M., and P. M. Cala. "Labeling of the Amphiuma erythrocyte K+/H+ exchanger with H2DIDS." American Journal of Physiology-Cell Physiology 267, no. 4 (October 1, 1994): C1002—C1012. http://dx.doi.org/10.1152/ajpcell.1994.267.4.c1002.

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Subsequent to swelling, the Amphiuma red blood cells lose K+, Cl-, and water until normal cell volume is restored. Net solute loss is the result of K+/H+ and Cl-/HCO3- exchangers functionally coupled through changes in pH and therefore HCO3-. Whereas the Cl-/HCO3- exchanger is constitutively active, K+/H+ actively is induced by cell swelling. The constitutive Cl-/HCO3- exchanger is inhibited by low concentrations (< 1 microM) of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or H2DIDS, yet the concentration of H2DIDS > 25 microM irreversibly modifies the K+/H+ exchanger in swollen cells. We exploited the volume-dependent irreversible low-affinity reaction between H2DIDS and the K+/H+ to identify the protein(s) associated with K+/H+ exchange activity. Labeling of the membrane proteins of intact cells with 3H2DIDS results in high-affinity labeling of a broad 100-kDa band, thought to be the anion exchanger. Additional swelling-dependent low-affinity labeling at 110 kDa suggests the possibility of a volume-induced population of anion exchangers. Finally, the correlation between volume-sensitive K+/H+ modification and low-affinity labeling suggests that transport activity is associated with a protein of approximately 85 kDa. Although a 55-kDa protein is also labeled, it is a less likely candidate, since label incorporation and transport modification are less well correlated than that of the 85- and 110-kDa proteins.

6

Chen, Xi, Fu Li, and Yao-Wen Wu. "Chemical labeling of intracellular proteins via affinity conjugation and strain-promoted cycloadditions in live cells." Chemical Communications 51, no. 92 (2015): 16537–40. http://dx.doi.org/10.1039/c5cc05208d.

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7

Masselin, Arnaud, Antoine Petrelli, Maxime Donzel, Sylvie Armand, Sylvain Cottaz, and Sébastien Fort. "Unprecedented Affinity Labeling of Carbohydrate-Binding Proteins with s-Triazinyl Glycosides." Bioconjugate Chemistry 30, no. 9 (August 12, 2019): 2332–39. http://dx.doi.org/10.1021/acs.bioconjchem.9b00432.

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8

Vale, M. G. "Affinity labeling of calmodulin-binding proteins in skeletal muscle sarcoplasmic reticulum." Journal of Biological Chemistry 263, no. 26 (September 1988): 12872–77. http://dx.doi.org/10.1016/s0021-9258(18)37642-7.

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9

Laudon, Moshe, and Nava Zisapel. "Melatonin binding proteins identified in the rat brain by affinity labeling." FEBS Letters 288, no. 1-2 (August 19, 1991): 105–8. http://dx.doi.org/10.1016/0014-5793(91)81013-x.

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10

Kuwahara, Daichi, Takahiro Hasumi, Hajime Kaneko, Madoka Unno, Daisuke Takahashi, and Kazunobu Toshima. "A solid-phase affinity labeling method for target-selective isolation and modification of proteins." Chem. Commun. 50, no. 98 (2014): 15601–4. http://dx.doi.org/10.1039/c4cc06783e.

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Solid-phase affinity labeling of target proteins with the specifically designed chemical tools selectively and effectively furnished the labeled target proteins without the need for tedious manipulations.

11

Cullen, Paul A., Xiaoyi Xu, James Matsunaga, Yolanda Sanchez, Albert I. Ko, David A. Haake, and Ben Adler. "Surfaceome of Leptospira spp." Infection and Immunity 73, no. 8 (August 2005): 4853–63. http://dx.doi.org/10.1128/iai.73.8.4853-4863.2005.

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ABSTRACT The identification of the subset of outer membrane proteins exposed on the surface of a bacterial cell (the surfaceome) is critical to understanding the interactions of bacteria with their environments and greatly narrows the search for protective antigens of extracellular pathogens. The surfaceome of Leptospira was investigated by biotin labeling of viable leptospires, affinity capture of the biotinylated proteins, two-dimensional gel electrophoresis, and mass spectrometry (MS). The leptospiral surfaceome was found to be predominantly made up of a small number of already characterized proteins, being in order of relative abundance on the cell surface: LipL32 > LipL21 > LipL41. Of these proteins, only LipL32 had not been previously identified as surface exposed. LipL32 surface exposure was subsequently verified by three independent approaches: surface immunofluorescence, whole-cell enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Three other proteins, Q8F8Q0 (a putative transmembrane outer membrane protein) and two proteins of 20 kDa and 55 kDa that could not be identified by MS, one of which demonstrated a high degree of labeling potentially representing an additional, as-yet-uncharacterized, surface-exposed protein. Minor labeling of p31LipL45, GroEL, and FlaB1 was also observed. Expression of the surfaceome constituents remained unchanged under a range of conditions investigated, including temperature and the presence of serum or urine. Immunization of mice with affinity-captured surface components stimulated the production of antibodies that bound surface proteins from heterologous leptospiral strains. The surfaceomics approach is particularly amenable to protein expression profiling using small amounts of sample (<107 cells) offering the potential to analyze bacterial surface expression during infection.

12

Cheng, Bo, Qi Tang, Che Zhang, and Xing Chen. "Glycan Labeling and Analysis in Cells and In Vivo." Annual Review of Analytical Chemistry 14, no. 1 (June 5, 2021): 363–87. http://dx.doi.org/10.1146/annurev-anchem-091620-091314.

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As one of the major types of biomacromolecules in the cell, glycans play essential functional roles in various biological processes. Compared with proteins and nucleic acids, the analysis of glycans in situ has been more challenging. Herein we review recent advances in the development of methods and strategies for labeling, imaging, and profiling of glycans in cells and in vivo. Cellular glycans can be labeled by affinity-based probes, including lectin and antibody conjugates, direct chemical modification, metabolic glycan labeling, and chemoenzymatic labeling. These methods have been applied to label glycans with fluorophores, which enables the visualization and tracking of glycans in cells, tissues, and living organisms. Alternatively, labeling glycans with affinity tags has enabled the enrichment of glycoproteins for glycoproteomic profiling. Built on the glycan labeling methods, strategies enabling cell-selective and tissue-specific glycan labeling and protein-specific glycan imaging have been developed. With these methods and strategies, researchers are now better poised than ever to dissect the biological function of glycans in physiological or pathological contexts.

13

Hayashi, Takahiro, and Itaru Hamachi. "Traceless Affinity Labeling of Endogenous Proteins for Functional Analysis in Living Cells." Accounts of Chemical Research 45, no. 9 (June 8, 2012): 1460–69. http://dx.doi.org/10.1021/ar200334r.

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14

Goshe, Michael B., Josip Blonder, and Richard D. Smith. "Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for Proteome-Wide Analysis." Journal of Proteome Research 2, no. 2 (April 2003): 153–61. http://dx.doi.org/10.1021/pr0255607.

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15

Zhang, Jianfu, Jianzhao Peng, Yiran Huang, Ling Meng, Qingrong Li, Feng Xiong, and Xiaoyu Li. "Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling." Angewandte Chemie International Edition 59, no. 40 (August 11, 2020): 17525–32. http://dx.doi.org/10.1002/anie.202001205.

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16

Zhang, Jianfu, Jianzhao Peng, Yiran Huang, Ling Meng, Qingrong Li, Feng Xiong, and Xiaoyu Li. "Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling." Angewandte Chemie 132, no. 40 (August 11, 2020): 17678–85. http://dx.doi.org/10.1002/ange.202001205.

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17

Cosma, Antonio. "Affinity Biotinylation: Nonradioactive Method for Specific Selection and Labeling of Cellular Proteins." Analytical Biochemistry 252, no. 1 (October 1997): 10–14. http://dx.doi.org/10.1006/abio.1997.2289.

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18

Weissinger, Ronja, Lisa Heinold, Saira Akram, Ralf-Peter Jansen, and Orit Hermesh. "RNA Proximity Labeling: A New Detection Tool for RNA–Protein Interactions." Molecules 26, no. 8 (April 14, 2021): 2270. http://dx.doi.org/10.3390/molecules26082270.

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Multiple cellular functions are controlled by the interaction of RNAs and proteins. Together with the RNAs they control, RNA interacting proteins form RNA protein complexes, which are considered to serve as the true regulatory units for post-transcriptional gene expression. To understand how RNAs are modified, transported, and regulated therefore requires specific knowledge of their interaction partners. To this end, multiple techniques have been developed to characterize the interaction between RNAs and proteins. In this review, we briefly summarize the common methods to study RNA–protein interaction including crosslinking and immunoprecipitation (CLIP), and aptamer- or antisense oligonucleotide-based RNA affinity purification. Following this, we focus on in vivo proximity labeling to study RNA–protein interactions. In proximity labeling, a labeling enzyme like ascorbate peroxidase or biotin ligase is targeted to specific RNAs, RNA-binding proteins, or even cellular compartments and uses biotin to label the proteins and RNAs in its vicinity. The tagged molecules are then enriched and analyzed by mass spectrometry or RNA-Seq. We highlight the latest studies that exemplify the strength of this approach for the characterization of RNA protein complexes and distribution of RNAs in vivo.

19

Robinson, M. S., and B. M. Pearse. "Immunofluorescent localization of 100K coated vesicle proteins." Journal of Cell Biology 102, no. 1 (January 1, 1986): 48–54. http://dx.doi.org/10.1083/jcb.102.1.48.

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A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.

20

Trinkle-Mulcahy, Laura. "Recent advances in proximity-based labeling methods for interactome mapping." F1000Research 8 (January 31, 2019): 135. http://dx.doi.org/10.12688/f1000research.16903.1.

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Proximity-based labeling has emerged as a powerful complementary approach to classic affinity purification of multiprotein complexes in the mapping of protein–protein interactions. Ongoing optimization of enzyme tags and delivery methods has improved both temporal and spatial resolution, and the technique has been successfully employed in numerous small-scale (single complex mapping) and large-scale (network mapping) initiatives. When paired with quantitative proteomic approaches, the ability of these assays to provide snapshots of stable and transient interactions over time greatly facilitates the mapping of dynamic interactomes. Furthermore, recent innovations have extended biotin-based proximity labeling techniques such as BioID and APEX beyond classic protein-centric assays (tag a protein to label neighboring proteins) to include RNA-centric (tag an RNA species to label RNA-binding proteins) and DNA-centric (tag a gene locus to label associated protein complexes) assays.

21

Braner, M., A. Kollmannsperger, R. Wieneke, and R. Tampé. "‘Traceless’ tracing of proteins – high-affinity trans-splicing directed by a minimal interaction pair." Chemical Science 7, no. 4 (2016): 2646–52. http://dx.doi.org/10.1039/c5sc02936h.

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Using a minimal lock-and-key element the affinity between the intein fragments for N-terminal protein trans-splicing was significantly increased, allowing for site-specific, ‘traceless’ covalent protein labeling in living mammalian cells at nanomolar probe concentrations.

22

Pfeuffer, Elke, and Thomas Pfeuffer. "Affinity labeling of forskolin-binding proteins comparison between glucose carrier and adenylate cyclase." FEBS Letters 248, no. 1-2 (May 8, 1989): 13–17. http://dx.doi.org/10.1016/0014-5793(89)80422-3.

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23

Tomohiro, Takenori, Hirotsugu Inoguchi, Souta Masuda, and Yasumaru Hatanaka. "Affinity-based fluorogenic labeling of ATP-binding proteins with sequential photoactivatable cross-linkers." Bioorganic & Medicinal Chemistry Letters 23, no. 20 (October 2013): 5605–8. http://dx.doi.org/10.1016/j.bmcl.2013.08.041.

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24

Konziase, Benetode. "Biotinylated probes of artemisinin with labeling affinity toward Trypanosoma brucei brucei target proteins." Analytical Biochemistry 482 (August 2015): 25–31. http://dx.doi.org/10.1016/j.ab.2015.04.020.

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25

Safer, B., R. B. Cohen, S. Garfinkel, and J. A. Thompson. "DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins." Molecular and Cellular Biology 8, no. 1 (January 1988): 105–13. http://dx.doi.org/10.1128/mcb.8.1.105.

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A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. 32P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of 32P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.

26

Safer, B., R. B. Cohen, S. Garfinkel, and J. A. Thompson. "DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins." Molecular and Cellular Biology 8, no. 1 (January 1988): 105–13. http://dx.doi.org/10.1128/mcb.8.1.105-113.1988.

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A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. 32P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of 32P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.

27

Wong, Franklin C., John Boja, Beng Ho, Michael J. Kuhar, and Dean F. Wong. "Affinity Labeling of Membrane Receptors Using Tissue-Penetrating Radiations." BioMed Research International 2013 (2013): 1–7. http://dx.doi.org/10.1155/2013/503095.

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Photoaffinity labeling, a usefulin vivobiochemical tool, is limited when appliedin vivobecause of the poor tissue penetration by ultraviolet (UV) photons. This study investigates affinity labeling using tissue-penetrating radiation to overcome the tissue attenuation and irreversibly label membrane receptor proteins. Using X-ray (115 kVp) at low doses (<50 cGy or Rad), specific and irreversible binding was found on striatal dopamine transporters with 3 photoaffinity ligands for dopamine transporters, to different extents. Upon X-ray exposure (115 kVp), RTI-38 and RTI-78 ligands showed irreversible and specific binding to the dopamine transporter similar to those seen with UV exposure under other conditions. Similarly, gamma rays at higher energy (662 keV) also affect irreversible binding of photoreactive ligands to peripheral benzodiazepine receptors (by PK14105) and to the dopamine (D2) membrane receptors (by azidoclebopride), respectively. This study reports that X-ray and gamma rays induced affinity labeling of membrane receptors in a manner similar to UV with photoreactive ligands of the dopamine transporter, D2 dopamine receptor (D2R), and peripheral benzodiazepine receptor (PBDZR). It may provide specific noninvasive irreversible block or stimulation of a receptor using tissue-penetrating radiation targeting selected anatomic sites.

28

Yefidoff, Revital, and Amnon Albeck. "12-Substituted-13,14-dihydroretinols designed for affinity labeling of retinol binding- and processing proteins." Tetrahedron 60, no. 37 (September 2004): 8093–102. http://dx.doi.org/10.1016/j.tet.2004.06.116.

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29

Fukui, Toshio. "Exploring the Nucleotide-Binding Site in Proteins by Affinity Labeling and Site-Directed Mutagenesis1." Journal of Biochemistry 117, no. 6 (June 1995): 1139–44. http://dx.doi.org/10.1093/oxfordjournals.jbchem.a124834.

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30

Gibson, Kathryn, Yumi Kumagai, and Yasuko Rikihisa. "Proteomic Analysis of Neorickettsia sennetsu Surface-Exposed Proteins and Porin Activity of the Major Surface Protein P51." Journal of Bacteriology 192, no. 22 (September 10, 2010): 5898–905. http://dx.doi.org/10.1128/jb.00632-10.

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ABSTRACT Neorickettsia sennetsu is an obligate intracellular bacterium of monocytes and macrophages and is the etiologic agent of human Sennetsu neorickettsiosis. Neorickettsia proteins expressed in mammalian host cells, including the surface proteins of Neorickettsia spp., have not been defined. In this paper, we isolated surface-exposed proteins from N. sennetsu by biotin surface labeling followed by streptavidin-affinity chromatography. Forty-two of the total of 936 (4.5%) N. sennetsu open reading frames (ORFs) were detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS), including six hypothetical proteins. Among the major proteins identified were the two major β-barrel proteins: the 51-kDa antigen (P51) and Neorickettsia surface protein 3 (Nsp3). Immunofluorescence labeling not only confirmed surface exposure of these proteins but also showed rosary-like circumferential labeling with anti-P51 for the majority of bacteria and polar to diffuse punctate labeling with anti-Nsp3 for a minority of bacteria. We found that the isolated outer membrane of N. sennetsu had porin activity, as measured by a proteoliposome swelling assay. This activity allowed the diffusion of l-glutamine, the monosaccharides arabinose and glucose, and the tetrasaccharide stachyose, which could be inhibited with anti-P51 antibody. We purified native P51 and Nsp3 under nondenaturing conditions. When reconstituted into proteoliposomes, purified P51, but not Nsp3, exhibited prominent porin activity. This the first proteomic study of a Neorickettsia sp. showing new sets of proteins evolved as major surface proteins for Neorickettsia and the first identification of a porin for the genus Neorickettsia.

31

Takata, K., and SJ Singer. "Localization of high concentrations of phosphotyrosine-modified proteins in mouse megakaryocytes." Blood 71, no. 3 (March 1, 1988): 818–21. http://dx.doi.org/10.1182/blood.v71.3.818.818.

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Abstract Phosphorylation of tyrosine residues of cellular proteins is a rare event and is considered to be related to the regulation of cellular growth, differentiation, and some forms of neoplastic transformation. Using high-affinity antibodies specific to phosphotyrosine (P-Tyr), we have shown the presence at high concentrations of P-Tyr-modified proteins in mouse bone-marrow megakaryocytes. Immunofluorescence microscopy of semithin frozen sections revealed that P-Tyr labeling was localized in a punctate pattern in the majority of the cytoplasm. The thin outer rim of the cytoplasm and the cell membrane was devoid of the label. Immunogold electron microscopy of ultrathin frozen sections showed that P-Tyr labeling was concentrated mostly on the membranes of the vesicles in the cytoplasm. The membrane demarcation system characteristic of megakaryocytes was not labeled. The intensity of P- Tyr labeling varied from one megakaryocyte to another. These results suggest that tyrosine phosphorylation of specific proteins might be correlated with the developmental stage of megakaryocytes, possibly related to the formation and deposition of the granules.

32

Takata, K., and SJ Singer. "Localization of high concentrations of phosphotyrosine-modified proteins in mouse megakaryocytes." Blood 71, no. 3 (March 1, 1988): 818–21. http://dx.doi.org/10.1182/blood.v71.3.818.bloodjournal713818.

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Phosphorylation of tyrosine residues of cellular proteins is a rare event and is considered to be related to the regulation of cellular growth, differentiation, and some forms of neoplastic transformation. Using high-affinity antibodies specific to phosphotyrosine (P-Tyr), we have shown the presence at high concentrations of P-Tyr-modified proteins in mouse bone-marrow megakaryocytes. Immunofluorescence microscopy of semithin frozen sections revealed that P-Tyr labeling was localized in a punctate pattern in the majority of the cytoplasm. The thin outer rim of the cytoplasm and the cell membrane was devoid of the label. Immunogold electron microscopy of ultrathin frozen sections showed that P-Tyr labeling was concentrated mostly on the membranes of the vesicles in the cytoplasm. The membrane demarcation system characteristic of megakaryocytes was not labeled. The intensity of P- Tyr labeling varied from one megakaryocyte to another. These results suggest that tyrosine phosphorylation of specific proteins might be correlated with the developmental stage of megakaryocytes, possibly related to the formation and deposition of the granules.

33

Keeble, Anthony H., Paula Turkki, Samuel Stokes, Irsyad N. A. Khairil Anuar, Rolle Rahikainen, Vesa P. Hytönen, and Mark Howarth. "Approaching infinite affinity through engineering of peptide–protein interaction." Proceedings of the National Academy of Sciences 116, no. 52 (December 10, 2019): 26523–33. http://dx.doi.org/10.1073/pnas.1909653116.

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Much of life’s complexity depends upon contacts between proteins with precise affinity and specificity. The successful application of engineered proteins often depends on high-stability binding to their target. In recent years, various approaches have enabled proteins to form irreversible covalent interactions with protein targets. However, the rate of such reactions is a major limitation to their use. Infinite affinity refers to the ideal where such covalent interaction occurs at the diffusion limit. Prototypes of infinite affinity pairs have been achieved using nonnatural reactive groups. After library-based evolution and rational design, here we establish a peptide–protein pair composed of the regular 20 amino acids that link together through an amide bond at a rate approaching the diffusion limit. Reaction occurs in a few minutes with both partners at low nanomolar concentration. Stopped flow fluorimetry illuminated the conformational dynamics involved in docking and reaction. Hydrogen–deuterium exchange mass spectrometry gave insight into the conformational flexibility of this split protein and the process of enhancing its reaction rate. We applied this reactive pair for specific labeling of a plasma membrane target in 1 min on live mammalian cells. Sensitive and specific detection was also confirmed by Western blot in a range of model organisms. The peptide–protein pair allowed reconstitution of a critical mechanotransmitter in the cytosol of mammalian cells, restoring cell adhesion and migration. This simple genetic encoding for rapid irreversible reaction should provide diverse opportunities to enhance protein function by rapid detection, stable anchoring, and multiplexing of protein functionality.

34

LeBel, Denis, та Marlyne Beattie. "Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[α-32P]ATP". Biochemistry and Cell Biology 64, № 1 (1 січня 1986): 13–20. http://dx.doi.org/10.1139/o86-003.

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Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[α-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 μM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 μM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A–Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-β-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.

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Mann, Jasdeep K., Daniel Demonte, Christopher M. Dundas, and Sheldon Park. "Cell labeling and proximity dependent biotinylation with engineered monomeric streptavidin." TECHNOLOGY 04, no. 03 (September 2016): 152–58. http://dx.doi.org/10.1142/s2339547816400057.

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Because streptavidin is a homotetramer, it can bind multiple biotinylated ligands and cause target aggregation. To allow biotin detection without clustering, we previously engineered monomeric streptavidin (mSA) that is structurally similar to a single streptavidin subunit. Introducing the S25H mutation near the binding site increases the biotin dissociation half-life t1/2 to 83 minutes. The slowly dissociating mutant, mSA2, is useful in imaging studies because it allows stable labeling of biotinylated targets. We show that mSA2 conjugated with Alexa 488 binds biotinylated receptors on HEK293 with high specificity, and bound mSA2–Alexa488 does not dissociate significantly during an imaging study lasting 50 minutes. As a structural monomer, mSA2 can be fused to other proteins to create bifunctional molecules. We tested the use of mSA2 in proximity dependent biotinylation, in which mSA2 is fused to a peptide or a protein that binds a protein of interest (POI) and is used to recruit photoactivatable biotin (PA-biotin) to the target molecule. Once the resulting cluster of interacting proteins is subjected to UV-initiated distance-dependent biotinylation, subsequent affinity purification of biotinylated proteins on streptavidin beads can identify protein molecules that interact with POI. In addition to proteins that directly interact with the mSA2 fusion, mSA2 also induces biotinylation of other proteins that are associated through a series of noncovalent interactions. We show that mSA2 fused to an antibody recognition domain can be recruited to the kinase Erk-2 using a commercially available antibody and induce biotinylation of a known Erk-2 substrate, GST-Elk-1. Therefore, mSA2 can be used to implement proximity dependent biotinylation and detect transient enzyme-substrate interactions.

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Takata, K., and S. J. Singer. "Phosphotyrosine-modified proteins are concentrated at the membranes of epithelial and endothelial cells during tissue development in chick embryos." Journal of Cell Biology 106, no. 5 (May 1, 1988): 1757–64. http://dx.doi.org/10.1083/jcb.106.5.1757.

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We have used high affinity polyclonal antibodies specific for phosphotyrosine (PTyr) residues to examine the localization in various chick embryonic tissues in situ of PTyr-modified proteins by immunocytochemical methods. During the period from 9 to 21 d of development, most tissues exhibit elevated levels of PTyr-modified proteins as determined by immunoblotting experiments of tissue extracts with the anti-PTyr antibodies (Maher, P. A., and E. B. Pasquale. 1988. J. Cell Biol. 106:1747-1755). By immunofluorescence labeling of semithin frozen sections, the highest concentrations of PTyr immunolabeling in all of the embryonic tissues examined were localized to the membranes of the epithelial and endothelial cells with other cells showing no detectable labeling. These results were confirmed by immunoelectron microscopic labeling, which showed particularly high concentrations of PTyr-modified proteins close to the membranes at the apical junctions. The corresponding adult tissues showed no labeling. It is proposed that these results reflect the molecular basis for the functional plasticity of epithelial and endothelial cell junctions during embryonic development.

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Choi, Woonyoung, Sonya W. Song, and Wei Zhang. "Understanding Cancer through Proteomics." Technology in Cancer Research & Treatment 1, no. 4 (August 2002): 221–30. http://dx.doi.org/10.1177/153303460200100402.

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Proteomics is a rapidly expanding discipline that aims to gain a comprehensive understanding of the expressions, modification, interactions, and regulation of proteins in cells. New high-throughput technologies, such as protein chips and isotope-coded affinity tag peptide labeling, coupled with classic technologies such as two-dimensional gel electrophoresis and mass spectrometry, complement genomic technologies, providing cancer researchers with powerful tools for cancer diagnosis and prognosis and for the identification of targets for therapy.

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Kerbler, Sandra M., Roberto Natale, Alisdair R. Fernie, and Youjun Zhang. "From Affinity to Proximity Techniques to Investigate Protein Complexes in Plants." International Journal of Molecular Sciences 22, no. 13 (July 1, 2021): 7101. http://dx.doi.org/10.3390/ijms22137101.

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The study of protein–protein interactions (PPIs) is fundamental in understanding the unique role of proteins within cells and their contribution to complex biological systems. While the toolkit to study PPIs has grown immensely in mammalian and unicellular eukaryote systems over recent years, application of these techniques in plants remains under-utilized. Affinity purification coupled to mass spectrometry (AP-MS) and proximity labeling coupled to mass spectrometry (PL-MS) are two powerful techniques that have significantly enhanced our understanding of PPIs. Relying on the specific binding properties of a protein to an immobilized ligand, AP is a fast, sensitive and targeted approach used to detect interactions between bait (protein of interest) and prey (interacting partners) under near-physiological conditions. Similarly, PL, which utilizes the close proximity of proteins to identify potential interacting partners, has the ability to detect transient or hydrophobic interactions under native conditions. Combined, these techniques have the potential to reveal an unprecedented spatial and temporal protein interaction network that better understands biological processes relevant to many fields of interest. In this review, we summarize the advantages and disadvantages of two increasingly common PPI determination techniques: AP-MS and PL-MS and discuss their important application to plant systems.

39

Ross, Gregory M., Brian E. McCarry, and Ram K. Mishra. "Covalent Affinity Labeling of Brain Catecholamine-Absorbing Proteins Using a High-Specific-Activity Substituted Tetrahydronaphthalene." Journal of Neurochemistry 65, no. 6 (November 23, 2002): 2783–89. http://dx.doi.org/10.1046/j.1471-4159.1995.65062783.x.

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40

Yang, Yin, Qing-Feng Li, Chan Cao, Feng Huang, and Xun-Cheng Su. "Site-Specific Labeling of Proteins with a Chemically Stable, High-Affinity Tag for Protein Study." Chemistry - A European Journal 19, no. 3 (November 14, 2012): 1097–103. http://dx.doi.org/10.1002/chem.201202495.

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41

Rickard, J. E., and T. E. Kreis. "Identification of a novel nucleotide-sensitive microtubule-binding protein in HeLa cells." Journal of Cell Biology 110, no. 5 (May 1, 1990): 1623–33. http://dx.doi.org/10.1083/jcb.110.5.1623.

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A protein of Mr 170,000 (170K protein) has been identified in HeLa cells, using an antiserum raised against HeLa nucleotide-sensitive microtubule-binding proteins. Affinity-purified antibodies specific for this 170K polypeptide were used for its characterization. In vitro sedimentation of the 170K protein with taxol microtubules polymerized from HeLa high-speed supernatant is enhanced in the presence of an ATP depleting system, but unaffected by the non-hydrolyzable ATP analogue AMP-PNP. In addition, it can be eluted from taxol microtubules by ATP or GTP, as well as NaCl. Thus it shows microtubule-binding characteristics distinct from those of the previously described classes of nucleotide-sensitive microtubule-binding proteins, the motor proteins kinesin and cytoplasmic dynein, homologues of which are also present in HeLa cells. The 170K protein sediments on sucrose gradients at approximately 6S, separate from kinesin (9.5S) and cytoplasmic dynein (20S), further indicating that it is not associated with these motor proteins. Immunofluorescence localization of the 170K protein shows a patchy distribution in interphase HeLa cells, often organized into linear arrays that correlate with microtubules. However, not all microtubules are labeled, and there is a significant accumulation of antigen at the peripheral ends of microtubules. In mitotic cells, 170K labeling is found in the spindle, but there is also dotty labeling in the cytoplasm. After depolymerization of microtubules by nocodazole, the staining pattern is also patchy but not organized in linear arrays, suggesting that the protein may be able to associate with other intracellular structures as well as microtubules. In vinblastine-treated cells, there is strong labeling of tubulin paracrystals, and random microtubules induced in vivo by taxol are also labeled by the antibodies. These immunofluorescence labeling patterns are stable to extraction of cells with Triton X-100 before fixation, further suggesting an association of the protein with cytoplasmic structures. In vivo, therefore, the 170K protein appears to be associated with a subset of microtubules at discrete sites. Its in vitro behavior suggests that it belongs to a novel class of nucleotide-sensitive microtubule-binding proteins.

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Hortin, GL. "Sulfation of tyrosine residues in coagulation factor V." Blood 76, no. 5 (September 1, 1990): 946–52. http://dx.doi.org/10.1182/blood.v76.5.946.946.

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Abstract Sulfation of human coagulation factor V was investigated by biosynthetically labeling the products of HepG2 cells with [35S]sulfate. There was abundant incorporation of the sulfate label into a product identified as factor V by immunoprecipitation, lability to proteases, affinity for the lectin jacalin, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two or more sites in factor V incorporated sulfate as indicated by labeling of different peptide chains of factor Va. The 150-Kd activation fragment of factor Va incorporated the greatest amounts of sulfate. This fragment of factor Va was bound selectively by jacalin-agarose, reflecting its content of O-linked oligosaccharides. Analysis of an alkaline hydrolysate of sulfate-labeled factor Va by anion-exchange chromatography showed that the sulfate occurred partly in tyrosine sulfate residues and partly in alkaline-labile linkages. Sulfate groups are potentially important structural and functional elements in factor V, and labeling with [35S]sulfate provides a useful approach for examining the biosynthesis and processing of this protein. The hypothesis is advanced that sites of sulfation in factor V and several other plasma proteins contribute to the affinity and specificity of thrombin for these molecules, just as it does for the interaction of thrombin with the potent inhibitor hirudin from leeches.

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Hortin, GL. "Sulfation of tyrosine residues in coagulation factor V." Blood 76, no. 5 (September 1, 1990): 946–52. http://dx.doi.org/10.1182/blood.v76.5.946.bloodjournal765946.

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Sulfation of human coagulation factor V was investigated by biosynthetically labeling the products of HepG2 cells with [35S]sulfate. There was abundant incorporation of the sulfate label into a product identified as factor V by immunoprecipitation, lability to proteases, affinity for the lectin jacalin, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two or more sites in factor V incorporated sulfate as indicated by labeling of different peptide chains of factor Va. The 150-Kd activation fragment of factor Va incorporated the greatest amounts of sulfate. This fragment of factor Va was bound selectively by jacalin-agarose, reflecting its content of O-linked oligosaccharides. Analysis of an alkaline hydrolysate of sulfate-labeled factor Va by anion-exchange chromatography showed that the sulfate occurred partly in tyrosine sulfate residues and partly in alkaline-labile linkages. Sulfate groups are potentially important structural and functional elements in factor V, and labeling with [35S]sulfate provides a useful approach for examining the biosynthesis and processing of this protein. The hypothesis is advanced that sites of sulfation in factor V and several other plasma proteins contribute to the affinity and specificity of thrombin for these molecules, just as it does for the interaction of thrombin with the potent inhibitor hirudin from leeches.

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Ye, Xian Zhi. "Application of Biological Target Fishing Technology in Drug Discovery." Materials Science Forum 980 (March 2020): 210–19. http://dx.doi.org/10.4028/www.scientific.net/msf.980.210.

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Target fishing, a cutting-edge technology for drug research and development, plays a significant role in drug discovery. Varieties of methods for finding small-molecule drug targets have come into being driven by genomics, proteomics, bioinformatics and other technologies. These new methods are mainly based on the expression of gene or protein and proteins properties, including affinity and stability and so on. A serious challenge for the most widely used small molecule drugs is the discovery and identification of biological (and potential therapeutic) targets. Herein, we enumerate five biological target fishing techniques, including surface plasma resonance (SPR) techniques, random photo modified probes, drug affinity responsive target stability, fishing-rod strategy, and photo affinity labeling. And then we introduces the principles of operation, practical applications in the biological field of five methods, and analysis of their shortcomings.

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Roux, Kyle J., Dae In Kim, Manfred Raida, and Brian Burke. "A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells." Journal of Cell Biology 196, no. 6 (March 12, 2012): 801–10. http://dx.doi.org/10.1083/jcb.201112098.

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We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment.

46

Luo, W., L. R. Latchney, and D. J. Culp. "G protein coupling to M1 and M3muscarinic receptors in sublingual glands." American Journal of Physiology-Cell Physiology 280, no. 4 (April 1, 2001): C884—C896. http://dx.doi.org/10.1152/ajpcell.2001.280.4.c884.

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Rat sublingual gland M1 and M3 muscarinic receptors each directly activate exocrine secretion. To investigate the functional role of coreceptor expression, we determined receptor-G protein coupling. Although membrane proteins of 40 and 41 kDa are ADP-ribosylated by pertussis toxin (PTX), and 44 kDa proteins by cholera toxin (CTX), both carbachol-stimulated high-affinity GTPase activity and the GTP-induced shift in agonist binding are insensitive to CTX or PTX. Carbachol enhances photoaffinity labeling ([α-32P]GTP-azidoaniline) of only 42-kDa proteins that are subsequently tractable to immunoprecipitation by antibodies specific for Gαq or Gα11 but not Gα12 or Gα13. Carbachol-stimulated photoaffinity labeling as well as phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis is reduced 55% and 60%, respectively, by M1 receptor blockade with m1-toxin. Gαq/11-specific antibody blocks carbachol-stimulated PIP2 hydrolysis. We also provide estimates of the molar ratios of receptors to Gαq and Gα11. Although simultaneous activation of M1 and M3receptors is required for a maximal response, both receptor subtypes are coupled to Gαq and Gα11 to stimulate exocrine secretion via redundant mechanisms.

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Pearson, R. K., E. M. Hadac, and L. J. Miller. "Structural analysis of a distinct subtype of CCK receptor on human gastric smooth muscle tumors." American Journal of Physiology-Gastrointestinal and Liver Physiology 256, no. 6 (June 1, 1989): G1005—G1010. http://dx.doi.org/10.1152/ajpgi.1989.256.6.g1005.

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Human gastric smooth muscle tumors (leiomyosarcomas) have been shown to express cholecystokinin (CCK) binding sites that are functionally similar to physiologically important receptors present on their cells of origin. In this work, we have applied affinity-labeling techniques using 125I-D-Tyr-Gly-[Nle28,31]CCK-(26-33) to attempt to define the ligand-binding subunit of this receptor, and we have used the receptor antagonist L364,718 and deglycosylating enzymes to compare this molecule with well-defined CCK receptors on the classical peripheral targets (pancreas and gallbladder) of this hormone. To validate the use of 125I-D-Tyr-Gly-[Nle28,31]CCK-(26-33) for this tissue, we demonstrated that it bound to leiomyosarcoma membranes in a rapid, reversible, saturable, specific, and high-affinity manner (Kd = 0.8 nM). Although previous affinity labeling of this tissue with a CCK-33-based probe identified multiple bands, only one of those candidate proteins was predominantly labeled in the present work (Mr 100,000) by using a probe that is cross-linked through a site in greater proximity to the receptor-binding domain. Labeling was inhibited in a concentration-dependent manner by CCK-8 but not by structurally unrelated ligands. Although endo-beta-N-acetyl-glucosaminidase F digestion shifted this band by Mr 5,000, demonstrating that it was a glycoprotein, the deglycosylation product was very different from other CCK receptors studied. Also, unlike pancreatic and gallbladder CCK receptors, affinity labeling of this receptor was not affected by L364,718. These observations confirm that the gastric smooth muscle tumor CCK receptor represents a receptor subtype that is distinct from other peripheral CCK receptors, biochemically as well as functionally.

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Haas, M., P. B. Dunham, and B. Forbush. "[3H]bumetanide binding to mouse kidney membranes: identification of corresponding membrane proteins." American Journal of Physiology-Cell Physiology 260, no. 4 (April 1, 1991): C791—C804. http://dx.doi.org/10.1152/ajpcell.1991.260.4.c791.

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Crude plasma membranes from whole mouse kidneys have two classes of [3H]bumetanide binding sites. High-affinity sites (K1/2 approximately equal to 0.04 microM; Bmax = 1-2 pmol/mg protein) are similar to those identified on dog kidney membranes (B. Forbush and H.C. Palfrey. J. Biol. Chem. 258: 11787-11792, 1983) both with respect to affinity and in that Na, K, and Cl are required for [3H]bumetanide binding. Low-affinity sites (K1/2 approximately equal to 1 microM; Bmax = 7-14 pmol/mg) are unaffected by removal of these ions; such sites are not seen with dog kidney. When mouse kidney membranes are photolabeled with 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid [( 3H]BSTBA), a photoreactive bumetanide analogue, specific incorporation of the label is seen in two regions. As with dog kidney [M. Haas and B. Forbush. Am. J. Physiol. 253 (Cell Physiol. 22): C243-C252, 1987], an approximately 150-kDa protein is labeled with high affinity (K1/2 approximately equal to 0.05 microM). This labeling also requires Na, K, and Cl and appears to correspond to the high-affinity [3H]bumetanide binding sites and to the Na-K-Cl cotransport system. A second peak of [3H]BSTBA photolabeling, centered at approximately 75 kDa, incorporates the label with lower affinity (K1/2 = 2-3 microM). The photolabeling at approximately 75 kDa is unaffected by Na, K, and Cl concentrations and thus may correspond, at least in part, to the low-affinity [3H]bumetanide binding sites. Western blot analysis of [3H]BSTBA-labeled mouse kidney membranes was performed using an antiserum raised to proteins of approximately 82 and approximately 39 kDa isolated from mouse Ehrlich ascites tumor cells using a bumetanide affinity gel (P. B. Dunham, F. Jessen, and E. K. Hoffmann. Proc. Natl. Acad. Sci. USA 87: 6828-6832, 1990). This antiserum cross-reacts with a approximately 150-kDa mouse kidney protein, the staining profile of which on Western blot corresponds very closely to the peak of specific [3H]BSTBA incorporation in this region. The antiserum also reacts with proteins in the range of 65-85 kDa, overlapping the low-affinity peak of [3H]BSTBA incorporation.

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Thibonnier, M., T. Goraya, and L. Berti-Mattera. "G protein coupling of human platelet V1 vascular vasopressin receptors." American Journal of Physiology-Cell Physiology 264, no. 5 (May 1, 1993): C1336—C1344. http://dx.doi.org/10.1152/ajpcell.1993.264.5.c1336.

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We used several approaches to identify the G protein coupled to V1 vascular arginine vasopressin (AVP) receptors of human platelets. In purified platelet membranes, high-affinity specific binding of [3H]AVP but not that of the V1 vascular antagonist [3H]d(CH2)5Tyr(Me)AVP was modulated by guanosine 5'-O-(3-thiotriphosphate) or sodium fluoride both in the presence and absence of MgCl2. AVP failed to modify the [alpha-32P]GTP labeling pattern or the cytosolic translocation of the 24- to 27-kDa GTP-binding proteins. AVP-stimulated GTPase activity of platelet membranes was blocked by antibodies specific for the COOH-terminal of the Gq alpha protein. AVP increased labeling of a 42-kDa platelet membrane protein by the photoreactive GTP analogue [alpha-32P]azidoanilido GTP. Immunoblotting of platelet proteins with various G protein-specific antibodies revealed that the 42-kDa protein labeled with [alpha-32P]azidoanilido GTP was immunoblotted only by antibodies specific for the alpha-subunit of GQ-11. Thus V1 vascular AVP receptors of human platelets are coupled in a divalent cation-dependent manner to a G protein belonging to the Gq-11 family.

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Kalkhof, Stefan, Stefan Schildbach, Conny Blumert, Friedemann Horn, Martin von Bergen, and Dirk Labudde. "PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data." BioMed Research International 2016 (2016): 1–13. http://dx.doi.org/10.1155/2016/2891918.

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The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6.

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