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Timnit Yosef Doctoral Dissertation1.pdf (4.55 MB)

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Clickable, Photoactive NAADP Analogs for Isolation and Purification of the Unknown NAADP Receptor.

Asfaha, Timnit Yosef
http://rave.ohiolink.edu/etdc/view?acc_num=mco1471643537

Abstract Details

2016, Doctor of Philosophy (PhD), University of Toledo, Medicinal Chemistry.
NAADP is the most potent calcium mobilizing second messenger known so far. It is shown to be crucial in different cellular functions. NAADP mobilizes calcium from intracellular stores and is best characterized in both mammals and echinoderms. Although several candidates have been considered as the NAADP binding protein over the years, the identity of NAADP binding protein is still under investigation and the mechanism of calcium signaling is still unclear. We propose that the isolation and characterization of this binding protein could provide valuable information on understanding the binding protein and the mechanisms involved. In an attempt to isolate the NAADP binding protein, we devised and synthesized several clickable photoactive bi-functional NAADP analogs to be used in photoaffinity labeling (PAL) experiments where the photoactive probe after irradiation covalently binds to the NAADP binding protein and the clickable group aids in the isolation and purification of the binding protein. Previous SAR studies have suggested that 5-nicotinyl and 8-adenosyl positions as structurally neutral positions for substitutions. Thus these two points were chosen as points of substitution for the synthesis of bi-functional NAADP analogs. Synthesis procedures utilized the pyridine-base exchange reaction catalyzed by Aplysia californica ADP-ribosyl cyclase to substitute an exogenous pyridine base for the nicotinamide in NADP (or in 8-N3-NADP) producing the NAADP analogs. These analogs were then assayed in sea urchin egg homogenates for their ability to inhibit 32P-NAADP binding in competition ligand binding experiments and for their ability to mobilize calcium from NAADP sensitive intracellular stores. Those NAADP analogs that were shown to bind at low concentration were utilized in photoaffinity labeling experiments. The photoactive NAADP analogs were tested for their ability to specifically covalently label NAADP binding proteins both in sea urchin and in human Jurkat cells. These complexes were also tested for their effectiveness at cross-linking to affinity tags and enable the isolation and purification of the unknown NAADP binding protein.
James Slama (Committee Chair)
Viranga Tillekeratne (Committee Member)
David Giovannucci (Committee Member)
177 p.
Biomedical Research; Chemistry; Organic Chemistry
Photoaffinity-labeling; NAADP; Click-reaction

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Citations

  • Asfaha, T. Y. (2016). Clickable, Photoactive NAADP Analogs for Isolation and Purification of the Unknown NAADP Receptor. [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1471643537

    APA Style (7th edition)

  • Asfaha, Timnit. Clickable, Photoactive NAADP Analogs for Isolation and Purification of the Unknown NAADP Receptor. 2016. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1471643537.

    MLA Style (8th edition)

  • Asfaha, Timnit. "Clickable, Photoactive NAADP Analogs for Isolation and Purification of the Unknown NAADP Receptor." Doctoral dissertation, University of Toledo, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=mco1471643537

    Chicago Manual of Style (17th edition)

mco1471643537
442
© 2016, all rights reserved.
This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.