title: Characterization of mammalian equilibrative nucleoside transporters (ENTs) by mass spectrometry.
abstract: Equilibrative nucleoside transporters (ENTs) are integral membrane proteins that facilitate the movement of nucleosides and hydrophilic nucleoside analog (NA) drugs across cell membranes. ENTs are also targets for cardioprotectant drugs, which block re-uptake of the purine nucleoside adenosine, thereby enhancing purinergic receptor signaling pathways. ENTs are therefore important contributors to drug bioavailability and efficacy. Despite this important clinical role, very little is known about the structure and regulation of ENTs. Biochemical and structural studies on ENT proteins have been limited by their low endogenous expression levels, hydrophobicity and labile nature. To address these issues, we developed an approach whereby tagged mammalian ENT1 protein was over-expressed in mammalian cell lines, confirmed to be functional and isolated by affinity purification to sufficient levels to be analyzed using MALDI-TOF and tandem MS mass spectrometry. This proteomic approach will allow for a more detailed analysis of the structure, function and regulation of ENTs in the future.
authors.affiliation: Department of Biology, York University, 4700 Keele St., Toronto, Ontario, Canada M3J 1P3.
authors.forename: German
authors.initials: G
authors.lastname: Reyes
authors.forename: Zlatina
authors.initials: Z
authors.lastname: Naydenova
authors.forename: Parween
authors.initials: P
authors.lastname: Abdulla
authors.forename: Maria
authors.initials: M
authors.lastname: Chalsev
authors.forename: Anita
authors.initials: A
authors.lastname: Villani
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Rose
authors.forename: Naz
authors.initials: N
authors.lastname: Chaudary
authors.forename: Leroi
authors.initials: L
authors.lastname: DeSouza
authors.forename: K W Michael
authors.initials: KW
authors.lastname: Siu
authors.forename: Imogen R
authors.initials: IR
authors.lastname: Coe
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D033705:Equilibrative Nucleoside Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009842:Oligopeptides; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D014357:Trypsin
chemical_list: D033705:Equilibrative Nucleoside Transport Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013868:Thioinosine; C085706:FLAG peptide; D014357:Trypsin; C001789:4-nitrobenzylthioinosine